TrkA antibodies and their applications

Abstract

The present disclosure provides TrkA antibodies, compositions comprising said antibodies, and methods of using said antibodies for the prevention and / or treatment of diseases or disorders associated with inappropriate expression or function of TrkA, such as pain.

Description

[Technical field]

[0001] TrkA antibody and its applications. [Background technology]

[0002] Neurotrophins are a family of peptide growth factors structurally related to the first member of the family, Nerve Growth Factor (NGF). Neurotrophins regulate neuronal differentiation and survival, as well as synaptic transmission, in both the peripheral and central nervous systems. In addition, NGF acts on a variety of non-neuronal tissues and cells, including immune system cells.

[0003] NGF acts through two membrane receptors present on target cells: the low-affinity p75 receptor and TrkA (tropomyosin receptor kinase A), a 140 kDa high-affinity transmembrane glycoprotein with tyrosine kinase activity. TrkA is expressed in neural crest neurons, sympathetic neurons, and cholinergic neurons of the basal forebrain and striatum, where it functions as a key mediator of NGF activity. TrkA is also expressed in some non-neuronal tissues and cells, including B lymphocytes.

[0004] Studies have shown a direct relationship between pain and the TrkA system, demonstrating the presence of mutations in the TrkA gene and the resulting absence of functional NGF receptors in four unrelated cases of chronic anesthesia of type 4 pain. Thus, the NGF-TrkA system provides a potential target for designing treatments for pain, i.e., treatments that can antagonize pain-related neuropathic syndromes via TrkA-effective antagonists.

[0005] Reported anti-TrkA antibodies often block the binding of NGF to TrkA, potentially causing undesirable side effects.

[0006] Therefore, there is a strong demand for the development of TrkA antagonists that have therapeutic effects but do not cause the above-mentioned side effects. Summary of the Invention

[0007] The present disclosure provides TrkA antibodies and uses thereof. In particular, the present disclosure provides anti-TrkA antibodies, compositions comprising said antibodies, and methods of using said antibodies for the treatment of pain, such as chronic pain of nociceptive, non-nociceptive, inflammatory, traumatic, neuropathic, nociplastic, or mixed etiology.

[0008] In one embodiment, the present disclosure provides a method for the specific binding of TrkA to a subject having a TrkA antibody that is capable of binding to TrkA and has a TrkA antibody binding capacity of about 5*10 as measured by Octet or SPR. -8 K under M D The present invention provides an antibody or antigen-binding fragment thereof that exhibits one or more properties selected from the group consisting of: being capable of binding to TrkA at a specific site in the cytoplasm, being capable of inhibiting NGF-induced activation of TrkA, not substantially blocking the binding of TrkA to NGF, not substantially competing with NGF for binding to TrkA, and being capable of reducing NGF-mediated pain sensitization. In particular, the antibody or antigen-binding fragment thereof of the present disclosure is capable of reducing NGF-mediated pain sensitization without substantially impairing the effect of NGF on neuronal growth and survival, i.e., is capable of selectively reducing NGF-mediated pain sensitization.

[0009] In some embodiments, the antibody or antigen-binding fragment thereof can recognize an epitope in the TrkA extracellular domain (ECD), the epitope comprising amino acid residues Q176, H178, G179, Q180, and P187 of SEQ ID NO:119.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof can specifically bind to Q176, H178, G179, Q180 and / or P187 of SEQ ID NO:119.

[0011] In some embodiments, the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, a fully human antibody, and a multispecific antibody (such as a bispecific antibody or a trispecific antibody).

[0012] In some embodiments, the antigen-binding fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab)2 fragment, an Fv fragment, a VHH, and a ScFv.

[0013] In some embodiments, the TrkA is human TrkA.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof can compete with a reference antibody for binding to TrkA, the reference antibody comprising light chain CDR1-3 and heavy chain CDR1-3, wherein the light chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 96-98, respectively, and the heavy chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 93-95, respectively.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof comprises at least one of light chain CDRs 1 to 3 of a light chain variable region, wherein the light chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 132, 134, 15, and 53.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof comprises at least one of heavy chain CDRs 1 to 3 of a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 131, 133, 14, and 51.

[0017] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain CDR1, wherein the light chain CDR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 96, 117, 9, and 50.

[0018] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain CDR1, wherein the light chain CDR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 9, 28, 50, 56, 80, and 88.

[0019] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain CDR2, wherein the light chain CDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 97, 118, and 11.

[0020] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain CDR2, wherein the light chain CDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 11, 30, 39, 58, 77, 82, 103, and 108.

[0021] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain CDR3, wherein the light chain CDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 98, 13, and 41.

[0022] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain CDR3, wherein the light chain CDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 13, 32, 41, 60.

[0023] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region, wherein the light chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 132, 134, 15, and 53.

[0024] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region, wherein the light chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 15, 18, 44, 53, 61, 78, 85, 90, 91, 100, 104, 107, and 113.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant region, wherein the light chain constant region comprises a human Igκ constant region or a human Igλ constant region.

[0026] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain CDR1, wherein the heavy chain CDR1 is SEQ ID NO: 93, 114 (SX 1 WX 2 Q, here X 1 is H or Y, and X 2is I or M), 1, and 47.

[0027] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain CDR1, wherein the heavy chain CDR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1, 20, 45, 47, 62, 101, and 109.

[0028] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain CDR2, wherein the heavy chain CDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 94, 115, 4, and 48.

[0029] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain CDR2, wherein the heavy chain CDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 4, 23, 46, 48, 65, 73, 75, 87, 102, and 110.

[0030] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain CDR3, wherein the heavy chain CDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 95, 116, 7, and 49.

[0031] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain CDR3, wherein the heavy chain CDR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 7, 26, 37, 49, 68, and 111.

[0032] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 131, 133, 14, and 51.

[0033] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 14, 16, 42, 51, 70, 74, 76, 84, 86, 89, 92, 99, 105, 106, and 112.

[0034] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region, wherein the heavy chain constant region comprises a human IgG constant region.

[0035] In some embodiments, the antibody or antigen-binding fragment thereof comprises: 1) light chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 96, 97, and 98, respectively, and heavy chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 93, 94, and 95, respectively; 2) light chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 117, 118, and 41, respectively, and heavy chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 114 (SX 1 WX 2 Q, here X 1 is H or Y, and X 2is I or M), 3) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 13, respectively, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 4, and 7, respectively, 4) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 28, 30, and 32, respectively, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 20, 23, and 26, respectively, 5) amino acid sequences set forth in SEQ ID NOs: 56, 58, and 60, respectively. and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 62, 65, and 68, respectively; 6) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 56, 58, and 60, respectively, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 20, 73, and 68, respectively; 7) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 56, 58, and 60, respectively, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 20, 75, and 68, respectively; 8) SEQ ID NO: 56, 7) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 56, 77, and 60, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 62, 65, and 68, respectively; 8) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 56, 77, and 60, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 20, 73, and 68, respectively; 9) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 56, 77, and 60, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 20, 73, and 68, respectively; 10) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 56, 77, and 60, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 20, 75, and 68, respectively. 10) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 80, 82, and 32, respectively, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 62, 79, and 26, respectively; 11) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 80, 82, and 32, respectively, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 62, 79, and 26, respectively; 12) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 88, 30, and 32, respectively, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 20, 87, and 26, respectively; 13) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 28, 39, and 41, respectively, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 45, 46,and 37, 14) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 88, 103, and 41, respectively, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 101, 102, and 37, respectively, 15) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 88, 103, and 41, respectively, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 109, 110, and 111, respectively, 16) amino acid sequences set forth in SEQ ID NOs: 88, 39, and 41, respectively. 17) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 39, 108, and 41, respectively, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 109, 110, and 111, respectively; or 18) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 50, 39, and 41, respectively, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 47, 48, and 49, respectively.

[0036] In some embodiments, the antibody or antigen-binding fragment thereof comprises: 1) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 132, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 131; 2) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 134, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 133; 3) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 15, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 14; 4) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 18, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16; 5) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 90, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 89; 6) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 91, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 86; 7) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 91, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 92; 8) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 61, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70; 10) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 61, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 74; 11) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 78, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70; 12) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 78, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 74; 13) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 78, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 76; 14) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 85, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 84; 15) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 44, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 42; 16) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 100, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 99; 17) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 104;and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 105, 18) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 104, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 112, 19) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 107, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 106, 20) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 113, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 112, or 21) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 53, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 51.

[0037] In another aspect, the disclosure provides a fusion protein comprising an antibody or antigen-binding fragment of the disclosure.

[0038] In another aspect, the present disclosure provides a protein complex comprising an antibody or antigen-binding fragment of this disclosure, or a fusion protein of this disclosure.

[0039] In another aspect, the disclosure provides one or more isolated nucleic acid molecules encoding an antibody or antigen-binding fragment of the disclosure, or a fusion protein of the disclosure.

[0040] In another aspect, the disclosure provides one or more vectors comprising one or more isolated nucleic acid molecules of the disclosure.

[0041] In another aspect, the disclosure provides a cell comprising an isolated nucleic acid molecule or vector of the disclosure.

[0042] In another aspect, the present disclosure provides a composition comprising an antibody or antigen-binding fragment, fusion protein, protein complex, isolated nucleic acid molecule, vector, and / or cell of the present disclosure, and optionally a pharma- ceutically acceptable excipient.

[0043] In some embodiments, the pharma- ceutically acceptable excipient in the composition comprises a buffer.

[0044] In some embodiments, the pH of the composition is about 1-13.

[0045] In another aspect, the invention provides the use of an antibody or antigen-binding fragment thereof, fusion protein, protein complex, isolated nucleic acid molecule, vector, cell, and / or composition of the invention in the manufacture of a medicament for the prevention and / or treatment of a disease or disorder associated with inappropriate expression or function of TrkA.

[0046] In some embodiments, the disease or disorder comprises pain.

[0047] In some embodiments, the pain comprises chronic pain.

[0048] In some embodiments, the disease or disorder comprises chronic pain due to nociceptive, inflammatory, neuropathic, proliferative, or mixed etiology.

[0049] In some embodiments, the disease or disorder comprises chronic pain resulting from a musculoskeletal or neurological disorder.

[0050] In some embodiments, the disease or disorder comprises post-operative pain, rheumatoid arthritis pain, neuropathic pain, and / or osteoarthritis pain.

[0051] In another aspect, the disclosure provides the use of an antibody or antigen-binding fragment thereof, a fusion protein, or a protein complex in the manufacture of a medicament for determining the presence and / or amount of TrkA in a sample.

[0052] In another aspect, the disclosure provides a method of producing an antibody or antigen-binding fragment thereof, or a fusion protein of the disclosure, the method comprising culturing a cell of the disclosure under conditions that allow expression of the antibody or antigen-binding fragment thereof, or the fusion protein.

[0053] In another aspect, the present disclosure provides a method of preventing and / or treating a disease or disorder in a subject in need thereof comprising administering to the subject an effective amount of an antibody or antigen-binding fragment, fusion protein, protein complex, isolated nucleic acid molecule, vector, cell, and / or composition of the present disclosure, wherein the disease or disorder is a disease or disorder associated with inappropriate expression or function of TrkA.

[0054] In some embodiments of this method, the disease or disorder comprises pain.

[0055] In some embodiments of this method, the pain comprises chronic pain.

[0056] In some embodiments of this method, the disease or disorder comprises chronic pain of nociceptive, inflammatory, neuropathic, proliferative, or mixed etiology.

[0057] In some embodiments of this method, the disease or disorder comprises chronic pain resulting from a musculoskeletal or neurological disorder.

[0058] In some embodiments of this method, the disease or disorder comprises post-operative pain, rheumatoid arthritis pain, neuropathic pain, and / or osteoarthritis pain.

[0059] In another aspect, the present disclosure provides a method for determining the presence and / or amount of TrkA in a sample, the method comprising: a) contacting the sample with an antibody or antigen-binding fragment, fusion protein, or protein complex of the disclosure; and b) determining the presence and / or amount of the antibody or antigen-binding fragment, fusion protein, or protein complex bound to the sample.

[0060] In another aspect, the disclosure provides an antibody or antigen-binding fragment thereof, fusion protein, protein complex, isolated nucleic acid molecule, vector, cell, or composition of the disclosure for a) preventing and / or treating a disease or disorder, and / or b) determining the presence and / or amount of TrkA in a sample, wherein the disease or disorder is a disease or disorder associated with inappropriate expression or function of TrkA.

[0061] In another aspect, the present disclosure provides a method for screening or obtaining a TrkA antibody that does not substantially inhibit binding of TrkA to NGF, comprising using an epitope of the TrkA extracellular domain (ECD) comprising amino acid residues Q176, H178, G179, Q180, and P187 of SEQ ID NO:119.

[0062] In some embodiments of this method, the TrkA antibody exhibits one or more properties selected from the group consisting of: about 5*10 -8 capable of binding to TrkA with a KD of less than M; capable of inhibiting NGF-induced activation of TrkA; not substantially competing with NGF for binding to TrkA; and capable of reducing pain sensitization, such as NGF-mediated pain sensitization (e.g., selectively reducing NGF-mediated pain sensitization without substantially impairing the effects of NGF on neuronal growth and survival).

[0063] In some embodiments, the method is an in vitro or ex vivo method.

[0064] In another aspect, the present disclosure provides the use of an epitope of the TrkA extracellular domain (ECD) in the manufacture of an agent for obtaining or screening a TrkA antibody that does not substantially inhibit binding of TrkA to NGF, wherein the epitope comprises amino acid residues Q176, H178, G179, Q180, and P187 of SEQ ID NO: 119.

[0065] In some embodiments of the use, the TrkA antibody exhibits one or more properties selected from the group consisting of: about 5*10 -8 capable of binding to TrkA with a KD of less than M; capable of inhibiting NGF-induced activation of TrkA; not substantially competing with NGF for binding to TrkA; and capable of reducing pain sensitization, such as NGF-mediated pain sensitization (e.g., selectively reducing NGF-mediated pain sensitization without substantially impairing the effects of NGF on neuronal growth and survival).

[0066] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, in which only exemplary embodiments of the present disclosure are shown and described. As will be understood, the present disclosure is capable of other and different embodiments, and several of its details are capable of modification in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0067] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief description of the drawings]

[0068] The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of the present disclosure will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "figures"), in which: [Figure 1] 1 shows the results of an ELISA binding analysis of exemplary antibodies of the disclosure. [Diagram 2] 1 shows the results of a FACS binding analysis of exemplary antibodies of the disclosure. [Figure 3A-3B]1 shows the results of NFAT functional analysis of exemplary antibodies of the disclosure. [Figure 4A-4B] 1 shows the binding and functional properties of exemplary antibodies of the disclosure. [Figure 5A-5B] 1 shows the binding and functional properties of exemplary antibodies of the disclosure. [Figure 6] 1 shows an epitope analysis of TrkA(ECD) and exemplary antibodies of the disclosure. [Figure 7A-7D] 1 shows the sensorgram of the TrkA antibody competition assay. [Figure 8A-8C] It is shown that the antibody of the present invention does not inhibit the binding between TrkA and NGF. [Figure 9] 1 shows the results of NFAT functional analysis of exemplary antibodies of the disclosure. [Figure 10] Bright field photographs taken 72 hours after DRG seeding are shown. [Figure 11] Bright field axon length statistics 72 hours after DRG seeding are shown. [Figure 12] 1 shows a scheme of intraplantar injection in an NGF-induced hypersensitivity model. [Figure 13A-13B] FIG. 1 shows a pharmacological assessment of NGF-induced mechanical and thermal sensitivity using exemplary antibodies of the disclosure. [Figure 14] FIG. 1 shows a schematic diagram of the formalin-induced pain test. [Figure 15A-15B] 4 shows the antinociceptive effect of an exemplary antibody of the invention in the formalin-induced pain test. [Figure 16] Schematic of osteoarthritis (OA) pain induced by MIA injection and mechanical hypersensitivity testing. [Figures 17A-17G] 1 shows the effect of an exemplary antibody of the disclosure on OA pain induced by MIA injection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0069] While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Various changes, modifications, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives can be used to the embodiments of the present invention described herein.

[0070] The term "antibody" as used herein generally refers to an immunoglobulin or immunoglobulin-like molecule capable of specifically recognizing or binding to an antigen. An antibody may comprise a light chain (L) and a heavy chain (H). The light chain of an antibody may be classified as kappa light chain and lambda light chain. The heavy chain is classified as μ, δ, γ, α or ε, and the antibody isotype is defined as IgM, IgD, IgG (such as IgG1, IgG2, IgG3 or IgG4 subtypes), IgA and IgE, respectively. Each heavy chain may comprise a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region may comprise three domains (CH1, CH2, and CH3). Each light chain may comprise a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region may comprise a CL domain. The VH and VL regions can also be further divided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy / light chain pair (VH and VL) form the antibody binding site, respectively. The distribution of amino acids into regions or domains follows the definitions of the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, (1987 and 1991)) or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917, Chothia et al. (1989) Nature 342:878-883. The term "antibody" is not intended to be limited by the method by which the antibody is produced. For example, it includes recombinant antibodies, monoclonal antibodies, and other forms of antibodies. In some cases, the antibodies of the present disclosure are isolated antibodies.

[0071] The term "antigen-binding fragment" as used herein generally refers to one or more fragments of a full-length antibody that retain the ability to bind to the same antigen (e.g., TrkA) that the antibody binds and / or competes with the intact antibody for antigen-specific binding. Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some cases, antigen-binding fragments include Fab, Fab', F(ab') and / or Fab'-binding fragments. 2 , F(ab) 2 , VHH, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single chain antibodies (e.g., ScFv), chimeric antibodies, diabodies, and polypeptides comprising at least a sufficient portion of an antibody to confer a particular antigen-binding ability to the polypeptide.

[0072] The term "TrkA" as used herein generally refers to high affinity nerve growth factor receptor, neurotrophic tyrosine kinase receptor type 1, TRK1 transforming tyrosine kinase protein, tropomyosin-related kinase A, tyrosine kinase receptor, tyrosine kinase receptor A, Trk-A, gp140trk, p140-TrkA, MTC, or TRK. TrkA is a receptor tyrosine kinase involved in the development and maturation of the central and peripheral nervous systems through the regulation of proliferation, differentiation, and survival of sympathetic and neural neurons. TrkA is a high affinity receptor for its primary ligand, NGF, and may also be activated by binding to NTF3 / neurotrophin-3. TrkA as used herein may include functional fragments, variants, isoforms, and homologous species of human TrkA. Thus, the antibodies of the present disclosure may cross-react with TrkA of species other than human, in some cases. In certain embodiments, the antibody is completely specific for one or more human TrkA proteins and may not exhibit species or other types of non-human cross-reactivity. The complete amino acid sequences of the four known human TrkA isoforms are found in UniProt / Swiss-Prot accession number P04629 (Consortium TU, (2012) Nucleic Acids Res. (Nucleic Acids Research)) 40 (D1): D71-D5). The four isoforms are generated by alternative splicing. Isoform TrkA-I is present in most non-neuronal tissues (UniProt / Swiss-Prot accession number P04629-2), isoform TrkA-II is expressed primarily in neural cells (UniProt / Swiss-Prot accession number P04629-1), and isoform TrkA-III is specifically expressed in multipotent neural stem and neural crest progenitor cells (UniProt / Swiss-Prot accession number P04629-4). A fourth isoform, which differs from isoform TrkA-II in residues 1-71 and is missing residues 393-398, is known as isoform 3 (UniProt / Swiss-Prot accession number P04629-3).The TrkA-II isoform is the major known isoform of TrkA. The isoform TrkA-I has enhanced responsiveness to the NTF3 neurotrophin, whereas the isoform TrkA-III is constitutively active and does not bind NGF.

[0073] As used herein, the term "biological activity of TrkA" or "biological activity of TrkA" generally refers to one or more of the following: the ability to bind to NGF or other neurotrophins; the ability to activate NGF-induced signaling pathways; the ability to promote cell differentiation, proliferation, survival, growth, migration, and other changes in cell physiology (in the case of neurons, including peripheral and central neurons, including changes in neuronal morphology, synapse formation, synaptic function, neurotransmitter and / or neuropeptide release and regeneration following injury); and the ability to mediate pain associated with bone metastasis and cancer pain.

[0074] The term "TrkA / NGF signaling pathway" as used herein generally refers to a signaling pathway associated with nerve growth factor (NGF) and tyrosine kinase A (TrkA). NGF peptides may engage the NGF pathway through TrkA phosphorylation, activation of ShcC / PI3K and Plc-γ / MAPK signaling, and promote AKT-dependent survival and CREB-driven neuronal activity. The TrkA / NGF signaling pathway may promote survival and innervation of sympathetic and sensory neurons, and may be associated with endocytosis and retrograde transport of NGF / TrkA-containing endosomes from axon terminals to cell bodies, for example, for activation of NGF-induced gene expression involved in neuronal survival and development.

[0075] The term "binding specificity" as used herein generally refers to the ability of a substance to specifically bind to another substance and not randomly bind to other substances. For example, a protein may specifically bind to another protein due to its specific structure. Binding specificity can be measured, for example, by cross-competition assay or other binding assays known in the art.

[0076] As used herein, "K D The term affinity constant generally refers to the dissociation constant. A dissociation constant is a specific type of equilibrium constant that measures the tendency of a larger entity to reversibly separate (dissociate) into smaller components, such as when a complex breaks down into its constituent molecules. The dissociation constant is the reciprocal of the association constant. In the specific case of an antibody (Ab) binding to an antigen (Ag), the term affinity constant usually refers to the association constant.

[0077] The term "K on " as used herein generally refers to the on-rate constant for a binding protein (e.g., an antibody or antigen-binding fragment thereof) to bind to an antigen to form a binding complex (e.g., an antibody / antigen complex). The term "K on " also refers to the "association rate constant" or "ka", which are used interchangeably herein. This value indicates the rate of binding of a binding protein to its target antigen, or the rate of complex formation between a binding protein (such as an antibody) and its corresponding antigen.

[0078] As used herein, "IC 50 The term "inhibitory effect" is commonly used to describe the median inhibitory concentration (IC 50 ), for example, refers to the ability of TrkA antibodies to inhibit NGF-induced TrkA activation.

[0079] The term "monoclonal antibody" as used herein generally refers to an antibody produced by the same immune cell that is all a clone of a unique parent cell. Monoclonal antibodies can have monovalent affinity, in that they bind to the same epitope (the part of an antigen recognized by the antibody). In some cases, monoclonal antibodies can have multispecificity, such as bispecificity or trispecificity. It has become an important tool in biochemistry, molecular biology, and medicine. Recently, several monoclonal antibody technologies have been developed, including phage display, single B cell culture, single cell amplification from various B cell populations, and single plasma cell testing techniques.

[0080] As used herein, the term "chimeric antibody" generally refers to an antibody in which the variable (V) regions of the light and heavy chains are of murine origin and the constant (C) regions are of human origin. Generally, chimeric antibodies retain the specificity and affinity of the original murine monoclonal antibody, but the HAMA response may be significantly reduced.

[0081] The term "humanized antibody" as used herein generally refers to an antibody from a non-human species whose protein sequence has been modified to enhance similarity to antibody variants naturally produced in humans. The amino acid sequence of a humanized antibody can be essentially identical to that of a human variant, even though some of the complementarity determining region (CDR) segments responsible for the ability of the antibody to bind to a target antigen are of non-human origin.

[0082] As used herein, the terms "fully human antibody" and "human antibody" are used interchangeably and generally refer to an antibody that comprises a human variable region, most preferably a human constant region. In certain embodiments, these terms refer to an antibody that comprises variable and constant regions of human origin. The term "fully human antibody" includes antibodies that have variable and constant regions that correspond to human germline immunoglobulin sequences, as described by Kabat et al. (See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., United States Department of Health and Human Services, NIH Publication No. 91-3242).

[0083] The term "Fab fragment" as used herein generally refers to a portion of an immunoglobulin molecule, such as an antigen-binding fragment. A Fab fragment may consist of a light chain and a portion of a heavy chain with a single antigen-binding site. A Fab fragment may be obtained by papain digestion of an immunoglobulin molecule. For example, a Fab fragment may consist of a constant domain and a variable domain for each of the heavy and light chains. The variable domain may contain a paratope (antigen-binding site) at the amino terminus of the immunoglobulin molecule that contains a set of complementarity determining regions. The enzyme papain may be used to cleave an immunoglobulin molecule into two Fab fragments and an Fc fragment. The enzyme pepsin cleaves below the hinge region, resulting in the F(ab') fragment. 2 A bivalent F(ab) fragment and a pFc' fragment are formed. 2 or F(ab') 2 The fragment contains two antigen-binding regions linked by disulfide bonds. F(ab) 2 or F(ab') 2 Reduction of the fragments produces two monovalent Fab or Fab' fragments with free sulfhydryl groups available for conjugation to other molecules.

[0084] The term "Fv fragment" as used herein generally refers to the smallest fragment generated from enzymatic cleavage of IgG and IgM class antibodies. Fv fragments contain an antigen-binding site consisting of the VH and VL domains, but lack the CH1 and CL domains. The VH and VL chains may be held together within the Fv fragment by non-covalent interactions.

[0085] The term "ScFv" as used herein generally refers to a single chain antibody fragment. ScFv is a recombinant single chain polypeptide molecule in which the light chain variable region and the heavy chain variable region of an antibody are linked directly or via a peptide linker. Single chain antibodies (ScFv) generally do not contain a portion of the Fc region of an antibody, but methods are known for adding the region to a known ScFv molecule as needed. See Helfrich et al., A rapid and versatile method for harnessing ScFv antibody fragments with various biological functions. J Immunol Methods 237:131-145 (2000) and de Haard et al., Creating and engineering human antibodies for immunotherapy. Advanced Drug Delivery Reviews 31:5-31 (1998).

[0086] The term "fusion protein," as used herein, generally refers to a polypeptide that comprises, or alternatively consists of, the amino acid sequence of a polypeptide fused directly or indirectly (e.g., via a linker) to the amino acid sequence of a heterologous polypeptide (i.e., a polypeptide of different origin, sequence or structure).

[0087] The term "protein conjugate," as used herein, generally refers to a conjugate comprising a protein (e.g., an antibody or functional fragment thereof) bound to one or more additional moieties, such as a cytotoxic agent, e.g., a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), a label (e.g., a fluorescent label), and / or a radioactive isotope (i.e., a radioconjugate).

[0088] A number of CDR definitions are in use and are included herein. The Kabat definition is based on sequence variability and is the most commonly used (Kabat EA et al., supra). The Chothia definition is based on the location of the structural loops (Chothia & Lesk J. (1987) Mol. Biol. 196:901-917). The AbM definition is a compromise between the Kabat and Chothia definitions and is used in Oxford Molecular's AbM antibody modeling software (Martin ACR et al., (1989) PNAS USA 86:9268-9272; Martin ACR et al., (1991) Methods Enzymol. 203:121-153; Pedersen JT et al., (1992) Immunomethods 1:126-136; Rees AR et al., (1996) Sternberg MJE (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172). The contact definition has been introduced recently (MacCallum RM et al. (1996) J. Mol. Biol. 262:732-745) and is based on the analysis of complex structures available in the Protein Databank.The IMGT®, International Immunoglobulin Genetics Information System® (http: / / www.imgt.org) CDR definitions are based on the IMGT numbering for all immunoglobulin and T-cell receptor V regions from all species (IMGT®, International Immunoglobulin Genetics Information System®, Lefranc MP et al., (1999) Nucleic Acids Res. 27(1):209-12; Ruiz M et al., (2000) Nucleic Acids Res. 28(1):219-21; Lefranc MP (2001) Nucleic Acids Res. 29(1):207-9; Lefranc MP (2003) Nucleic Acids Res. 31(1):307-10; Lefranc MP et al. (2005) Dev. Comp. Immunol. 29(3):185-203; Kaas Q et al., (2007) Briefings in Functional Genomics&Proteomics, 6(4):253-64).

[0089] The term "isolated nucleic acid molecule," as used herein, generally refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, that have been isolated from a natural environment or synthesized artificially.

[0090] The term "vector" as used herein generally refers to a nucleic acid vehicle into which a polynucleotide encoding a protein can be inserted and expressed. The genetic material elements carried by the vector can be expressed in a host cell by transforming, transducing, or transfecting the host cell with the vector. A vector may contain various elements that control expression, such as promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain an origin of replication. A vector may also contain components that aid in entry into a cell, such as, but are not limited to, viral particles, liposomes, and protein shells.

[0091] The term "cell" as used herein generally refers to a cell that can be used to carry a vector of the invention or to express or produce an antibody, antigen-binding fragment of the invention. The cell of the present disclosure may be a host cell.

[0092] As used herein, the terms "disease" and "disorder" are used interchangeably and generally refer to any condition that impairs the normal functioning of the body. A disease is often interpreted as a medical condition with specific symptoms or signs. It may be caused by external factors, such as pathogens, or by internal malfunctions of the immune system, such as immunodeficiency, or hypersensitivity, such as allergies or autoimmunity.

[0093] The term "subject" as used herein includes any human or non-human animal. The term "non-human animal" includes all vertebrates, including mammals and non-mammals, such as primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. For example, the subject may be a human.

[0094] The term "effective amount" as used herein generally refers to a dosage that is sufficient to provide a sufficient concentration to give beneficial effects to the recipient.The specific therapeutically effective dosage level for a specific subject varies depending on various factors, including the disease or disorder to be treated, the severity of the disease or disorder, the activity of the specific component, the route of administration, clearance rate, duration of treatment, the age, weight, sex, diet, and general health of the subject, and other related factors.

[0095] As used herein, the term "pharmaceutically acceptable excipient" generally refers to any solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

[0096] The term "about" as used herein generally refers to an approximation to a given value that is reasonably expected based on ordinary skill in the art, and includes equivalent and approximation values ​​based on experimental and / or measurement conditions for such a given value. For example, it may refer to values ​​above or below the value modified by the term by within 10%.

[0097] The term "polypeptide" or "protein" as used herein generally refers to a polymer having the amino acid sequence of a native protein, i.e., a protein produced by naturally occurring non-recombinant cells, or a molecule produced by genetically engineered or recombinant cells and having the amino acid sequence of a native protein, or a molecule having deletions, additions, and / or substitutions of one or more amino acids of the native sequence. The term also includes amino acid polymers in which one or more amino acids are chemical analogs of the corresponding native amino acids and polymers. The terms "polypeptide" and "protein" specifically encompass TrkA antigen binding proteins, antibodies, or sequences having deletions, additions, and / or substitutions of one or more amino acids of the antigen binding protein. The term "polypeptide fragment" refers to a polypeptide having an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion compared to the full-length native protein. Such fragments may also include modified amino acids compared to the native protein. In certain embodiments, the fragments are about 5-500 amino acids in length. For example, the fragments will be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length. Useful polypeptide fragments include immunologically functional fragments of antibodies that contain the binding domain. In the case of TrkA-binding antibodies, useful fragments include, but are not limited to, the CDR regions, the variable domains of the heavy and / or light chains, a portion of the antibody chain, or only the variable regions thereof that contain two CDRs.

[0098] The term "isolated protein" (such as an isolated antibody) as used herein generally refers to a protein of interest that is (1) free from at least a portion of other proteins with which it is normally associated, (2) essentially free from other proteins from the same source, e.g., the same species, (3) expressed by cells of a different species, (4) separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated, (5) functionally associated (by covalent or non-covalent interactions) with polypeptides with which it is not naturally associated, or (6) not occurring in nature. Typically, an "isolated protein" comprises at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. Genomic DNA, cDNA, mRNA or other RNA of synthetic origin, or any combination thereof, can encode such an isolated protein. Preferably, an isolated protein is substantially free of proteins or polypeptides or other contaminants found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic, research, or other uses.

[0099] A "variant" of a polypeptide (e.g., an antigen binding protein or antibody) comprises an amino acid sequence in which one or more amino acid residues have been inserted, deleted and / or substituted compared to another polypeptide sequence. Variants include fusion proteins.

[0100] The term "identity" as used herein generally refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent identity" refers to the percentage of identical residues between amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest molecule being compared. In these calculations, alignment gaps (if any) are preferably addressed by a specific mathematical model or computer program (i.e., "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology (Lesk, A.M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects (Smith, D.W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A.M. and Griffin, H.G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al, 1988, SIAM J. Applied Math. 48:1073.

[0101] When calculating percent identity, the sequences being compared are usually aligned to maximize the match between the sequences. One example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387, Genetics Computer Group, University of Wisconsin, Madison, WI). The computer algorithm GAP is used to align two polypeptides or polynucleotides for which the percent sequence identity is to be determined. The sequences are aligned so that their respective amino acids or nucleotides are best matched (the "area of ​​match" determined by the algorithm). A comparison matrix such as PAM 250 or BLOSUM 62 is used in conjunction with the algorithm, in addition to the gap opening penalty (calculated as 3 times the average diagonal, where the "average diagonal" is the average of the diagonals of the comparison matrix being used. The "diagonal" is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and gap extension penalty (usually 1 / 10 times the gap opening penalty). In certain embodiments, standard comparison matrices (for the PAM 250 comparison matrix, see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352; Henikoff et al., 1992, Proc. Natl. Acad. ScL USA 89:10915-10919 for the BLOSUM 62 comparison matrix) are also used in the algorithm.

[0102] Conservative amino acid substitutions may include non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than synthesis in biological systems, and include peptidomimetics and other reversed or inverted forms of amino acid moieties.

[0103] The "Fc" region is the C region of an antibody. H1 Domain and C H2It is composed of two heavy chain fragments that contain the C domain. The two heavy chain fragments are separated by two or more disulfide bonds and H3 The domains are held together by hydrophobic interactions.

[0104] A "multispecific antigen-binding protein" or "multispecific antibody" is a protein that targets multiple antigens or epitopes.

[0105] A "bispecific", "dual-specific" or "bifunctional" antigen-binding protein or antibody is a hybrid antigen-binding protein or antibody, respectively, that has two different antigen-binding sites. Bispecific antigen-binding proteins and antibodies are a type of multispecific antigen-binding protein antibody, and can be generated by a variety of methods, including fusion of hybridomas and binding of Fab' fragments. See, e.g., Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321; Kostelny et al., 1992, J. Immunol. 148:1547-1553. The two binding sites of a bispecific antigen-binding protein or antibody bind to two different epitopes present on the same or different protein targets.

[0106] The antigen-binding protein has a dissociation constant (Ka) of 10 -7 An antigen-binding protein is said to "specifically bind" to a target antigen when its K is less than M. D 5×10 -8 It specifically binds to an antigen with "high affinity" when its K is less than M, and D is 1×10 -8 A substance that specifically binds to an antigen with "very high affinity" when its M is less than M

[0107] An antigen-binding protein is "selective" if its binding to one target is stronger than its binding to other targets.

[0108] "Antigen-binding region" or "antigen-binding fragment" refers to a protein or a portion of a protein that specifically binds to a particular antigen (e.g., a paratope). For example, a portion of an antigen-binding protein that contains amino acid residues that interact with an antigen and confer specificity and affinity to the antigen-binding protein for the antigen is called an "antigen-binding region." An antigen-binding region typically includes one or more "complementary binding regions" ("CDRs"). A "CDR" is an amino acid sequence that contributes to antigen-binding specificity and affinity.

[0109] Unless otherwise specified, the term "antibody" includes antibodies that contain two full-length heavy chains and two full-length light chains, as well as their derivatives, variants, fragments, and muteins, examples of which are described below. Furthermore, unless expressly excluded, antibodies include monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and fragments thereof, respectively. In some embodiments, the term also includes peptibodies.

[0110] In certain embodiments, an antibody heavy chain binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody light chain binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an individual variable region specifically binds to an antigen in the absence of other variable regions.

[0111] In certain embodiments, a clear delineation of CDRs and identification of the residues that constitute the antibody binding site are achieved by solving the structure of the antibody and / or solving the structure of the antibody-ligand complex. In certain embodiments, this can be achieved by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, various analytical methods are employed to identify or approximate the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, the contact definition, and the like.

[0112] The term "competition," when used in the context of antigen binding proteins (such as antigen binding proteins or antibodies) competing for the same epitope, refers to competition between the antigen binding proteins as determined by an assay in which the antigen binding protein being tested (such as an antibody or an immunologically functional fragment thereof) prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (such as a ligand or reference antibody) to a common antigen (such as TrkA or a fragment thereof, e.g., its ECD). Many types of competitive binding assays can be used to determine whether one antigen binding protein competes with another antigen binding protein. For example, solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competitive assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253), solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid-phase direct label assays, solid-phase direct label sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press), solid-phase direct label RIA using 1-125 labels (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15), solid-phase direct biotin-avidin EIA (see, e.g., Cheung et al., 1990, Virology 176:546-552), and direct labeling RIA (see Moldenhauer et al, 1990, Scand. J. Immunol. 32:77-82). Typically, such assays use purified antigen bound to a solid surface, or cells containing either of these, an unlabeled test antigen-binding protein, and a labeled reference antigen-binding protein. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen-binding protein. Typically, the test antigen-binding protein is present in excess.Antigen binding proteins identified by competitive assays (competitor antigen binding proteins) include antigen binding proteins that bind to the same epitope as the reference antigen binding protein, and antigen binding proteins that bind to adjacent epitopes close enough to the epitope bound by the reference antigen binding protein that steric hindrance occurs. Typically, when a competing antigen binding protein is present in excess, specific binding of the reference antigen binding protein to a common antigen is inhibited (e.g., reduced) by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0113] The term "antigen" as used herein generally refers to a molecule or a portion of a molecule that can be bound by a selective binding agent, such as an antigen binding protein (including, for example, an antibody or an immunologically functional fragment thereof). In some embodiments, an antigen can be used in an animal to generate antibodies capable of binding to the antigen. An antigen can have one or more epitopes that can interact with different antigen binding proteins, such as antibodies.

[0114] The term "epitope" includes any determinant that can bind to an antigen-binding protein, such as an antibody or a T-cell receptor. An epitope is a region of an antigen to which an antigen-binding protein that targets that antigen binds, and, if the antigen is a protein, includes specific amino acids that are in direct contact with the antigen-binding protein. Most often, epitopes are found on proteins, but occasionally they may be found on other types of molecules, such as nucleic acids. Epitope determinants include chemically active surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl groups, and sulfonyl groups, and may have specific three-dimensional structural characteristics and specific charge characteristics. In general, an antibody specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and macromolecules.

[0115] As used herein, "substantially" or "substantially" generally means to a great or very large extent (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more).

[0116] The term "agent" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological material.

[0117] The terms "treat" and "treatment" include therapeutic treatments, prophylactic treatments, and applications in which a subject reduces the risk of developing a disorder or other risk factor. Treatment does not require a complete cure of a disease, but encompasses embodiments in which symptoms or underlying risk factors are alleviated.

[0118] The term "prevent" does not require 100% elimination of the possibility of an event, but rather indicates a reduced likelihood of the event occurring in the presence of a compound or method.

[0119] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly practiced in the art or as described herein. The techniques and procedures described above can generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and described throughout the specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), which is incorporated herein by reference for all purposes. Unless specific definitions are provided, the nomenclature used in connection with analytical chemistry, organic synthetic chemistry, medicinal chemistry and pharmaceutical chemistry described herein, as well as the laboratory procedures and techniques thereof, are those well known and commonly used in the art. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, administration, and treatment of patients.

[0120] Antibodies or antigen-binding fragments thereof In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof that binds to TrkA.

[0121] The antibody or antigen-binding fragment thereof can specifically bind to TrkA and does not substantially bind to tropomyosin receptor kinase B (TrkB), tropomyosin receptor kinase C (TrkC), or the p75 receptor.

[0122] The antibody or antigen-binding fragment has a binding affinity of approximately 5.0 × 10 as measured by Octet or SPR. -8 K under M D For example, K D is about 4.5 x 10 -8 Less than M, approx. 4 x 10 -8Less than M, approx. 3.5 x 10 -8 Less than M, about 3 x 10 -8 Less than M, approx. 2.8 x 10 -8 Less than M, approx. 2.7 x 10 -8 Less than M, approx. 2.5 x 10 -8 Less than M, about 2 x 10 -8 Less than M, approx. 1.5 x 10 -8 Less than M, approx. 1×10 -8 Less than M, approx. 8 x 10 -9 Less than M, about 5 x 10 -9 Less than M, about 5 x 10 -9 Less than M, approx. 4.5 x 10 -9 Less than M, approx. 4 x 10 -9 Less than M, approx. 3.5 x 10 -9 Less than M, about 3 x 10 -9 Less than M, approx. 2.5 x 10 -9 Less than M, about 2 x 10 -9 Less than M, approx. 1.5 x 10 -9 Less than M, approx. 1×10 -9 Less than M, approx. 1×10 -10 Less than M, approx. 1×10 -11 Less than M or about 1×10 -12 K less than M or even smaller D value.

[0123] The antibody, or antigen-binding fragment thereof, has a titer of approximately 1.5 x 10 as measured by Octet or SPR. 5 K exceeds (1 / Ms) on can bind to TrkA at, for example, about 2×10 5 (1 / Ms), about 3×10 5 (1 / Ms), about 4×10 5 (1 / Ms), about 5×10 5 (1 / Ms), about 6×10 5 (1 / Ms), about 7×10 5 (1 / Ms), about 8×10 5 (1 / Ms), about 9×10 5 (1 / Ms), approximately 1×10 6 K greater than or equal to (1 / Ms) on can bind to TrkA at

[0124] The antibody or antigen-binding fragment thereof can inhibit the TrkA / NGF signaling pathway. For example, the antibody or antigen-binding fragment thereof can inhibit NGF-induced activation of TrkA.

[0125] The antibody or antigen-binding fragment thereof may not substantially inhibit the binding between TrkA and NGF.

[0126] The antibody or antigen-binding fragment thereof may not substantially compete with NGF for binding to TrkA.

[0127] The antibody or antigen-binding fragment thereof can block NGF-mediated pain signaling.

[0128] The antibody or antigen-binding fragment thereof can reduce NGF-mediated mechanical and / or thermal sensitivity.

[0129] The antibody or antigen-binding fragment thereof is capable of inducing an antinociceptive effect in the formalin test.

[0130] This antibody or antigen-binding fragment thereof can ameliorate mechanical hypersensitivity in osteoarthritis pain induced by MIA injection.

[0131] The antibody or antigen-binding fragment thereof may be able to selectively attenuate NGF-mediated pain sensitization without substantially compromising the effects of NGF on neuronal growth and survival.

[0132] TrkA is also known as high affinity nerve growth factor receptor, neurotrophic tyrosine kinase receptor type 1, or TRK1 converting tyrosine kinase protein, and is a protein encoded by NTRK1 gene in humans. An exemplary human TrkA amino acid sequence is as set forth in SEQ ID NO: 119. As described herein, TrkA protein may also include fragments of full-length TrkA protein, such as its extracellular domain (ECD). An exemplary human TrkA ECD amino acid sequence is as set forth in SEQ ID NO: 120.

[0133] In some embodiments, an antibody or antigen-binding fragment thereof comprises one or more CDRs (e.g., 1, 2, 3, 4, 5, or 6 CDRs). In some embodiments, an antibody or antigen-binding fragment thereof comprises (a) a polypeptide structure and (b) one or more CDRs inserted and / or attached to the polypeptide structure. The polypeptide structure can take a variety of forms. For example, it can be or comprise the framework of a naturally occurring antibody, or a fragment or variant thereof, or it can be entirely synthetic.

[0134] In certain embodiments, the polypeptide structure of an antibody, or antigen-binding fragment thereof, is an antibody or is derived from an antibody, including, but not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as "antibody conjugates"), and portions or fragments of each. In some cases, an antibody or antigen-binding fragment thereof may be an immunological fragment of an antibody (e.g., Fab, Fab', F(ab')). 2 , or ScFv).

[0135] The antibody or antigen-binding fragment thereof may comprise a light chain constant region. The light chain constant region may comprise a human Igκ constant region or a human Igλ constant region. In some embodiments, the light chain constant region may comprise a human Igκ constant region. In some embodiments, the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO:121.

[0136] The antibody or antigen-binding fragment thereof may comprise a heavy chain constant region. The heavy chain constant region may comprise a human IgG constant region (such as a human IgG1, IgG2, or IgG4 constant region). In some embodiments, the heavy chain constant region comprises a human IgG4 constant region. In some embodiments, the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 122.

[0137] The variable regions of immunoglobulin chains generally exhibit the same overall structure, consisting of relatively conserved framework regions (FRs) combined with three hypervariable regions, more commonly called "complementarity determining regions" or CDRs. The CDRs of the two chains of each heavy / light chain pair mentioned above are usually aligned by the framework regions to form a structure that specifically binds to a particular epitope on a target protein (e.g., TrkA). From the N-terminus to the C-terminus, both naturally occurring light and heavy chain variable regions usually follow the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. A numbering system has been devised to assign numbers to the amino acids that occupy positions within each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, National Institutes of Health, Bethesda, MD), or Chothia & Lesk, 1987, J. MoL Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883.

[0138] Various heavy and light chain variable regions are provided herein. In some embodiments, each of these variable regions can be combined with a heavy chain constant region and a light chain constant region to form a complete antibody heavy chain and light chain, respectively. Furthermore, each of the heavy and light chain sequences thus generated can be combined to form a complete antibody structure.

[0139] Some specific examples of antibody light chain (VL) and heavy chain (VH) variable regions are provided and their corresponding amino acid sequences are summarized in Table 1 below.

[0140] [Table 1] Each of the exemplary variable heavy chains listed in Table 1 can be combined with any of the exemplary variable light chains listed in Table 1 to form an antibody. Table 1 shows exemplary light and heavy chain pairs found in some of the antibodies disclosed herein. In some cases, the antibody includes at least one variable heavy chain and one variable light chain of those listed in Table 1. In other examples, the antibody includes two identical light chains and two identical heavy chains. By way of example, the antibody or antigen-binding fragment thereof may include a heavy chain and a light chain, two heavy chains, or two light chains. In some embodiments, the antibody or antigen-binding fragment thereof includes (and / or consists of) one, two, and / or three heavy and / or light chain CDRs from at least one of the sequences listed in Table 1. In some embodiments, all six CDRs (CDR1-3 of the light chain (LCDR1, LCDR2, LCDR3) and CDR1-3 of the heavy chain (HCDR1, HCDR2, and HCDR3)) are part of the antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises one, two, three, four, five or more CDRs. In some embodiments, the antibody or antigen-binding fragment thereof comprises one heavy chain CDR and one light chain CDR of the CDRs in the sequence of Table 1. In some embodiments, the antibody or antigen-binding fragment thereof also comprises additional sections. Any light chain variable sequence (including CDR1, CDR2, and CDR3) can be selected from SEQ ID NOs: 132, 134, 15, and 53 below. Any heavy chain variable sequence (including CDR1, CDR2, and CDR3) can be selected from SEQ ID NOs: 131, 133, 14, and 51 below.

[0141] In some embodiments, the antibody or antigen-binding fragment thereof comprises (and / or consists of) an LCDR1 and an LCDR3 from at least one of the sequences listed in Table 1.

[0142] In some embodiments, the antibody or antigen-binding fragment thereof comprises (and / or consists of) an HCDR1 and an HCDR3 from at least one of the sequences listed in Table 1.

[0143] In some embodiments, the antibody or antigen-binding fragment thereof comprises (and / or consists of) an LCDR1, an LCDR3, an HCDR1 and an HCDR3 from at least one of the sequences listed in Table 1 (e.g., SEQ ID NOs: 91 and 86).

[0144] Examples of CDRs (determined according to the Kabat method) of the antibodies shown in Table 1 are shown in Table 2 below.

[0145] [Table 2] Some of the exemplary antibodies provided in this disclosure are considered to be alternatives or variants of each other. For example, muPHD48, PHD48-01, PHD48, PHD48-08, PHD22, PHD24, PHD25, PHD26, PHD28, PHD29, and PHD30 are considered to be alternatives or variants of each other. As another example, muPHD49, PHD49-01, PHD49-05, PHD49, PHD49-11, and PHD49-21 are considered to be alternatives or variants of each other.

[0146] In some embodiments, the antibody or antigen-binding fragment thereof can bind to an epitope recognized by one of the antibodies set forth in Table 1. In some embodiments, the antibody or antigen-binding fragment thereof can bind to a particular conformational state of TrkA and inhibit activation of TrkA (e.g., NGF-induced activation of TrkA) without blocking binding of NGF to TrkA.

[0147] As described herein, the TrkA antibody or antigen-binding fragment thereof can include a humanized antibody and / or a portion thereof. An important practical application of such a strategy is the "humanization" of the mouse humoral immune system. In certain embodiments, the humanized antibody is substantially non-immunogenic in humans. In certain embodiments, the humanized antibody has substantially the same affinity for a target as an antibody from another species from which the humanized antibody is derived.

[0148] In certain embodiments, amino acids in antibody variable domains are identified that can be modified without reducing the natural affinity of the antigen-binding domain and without reducing its immunogenicity.

[0149] In certain embodiments, antibody modification by methods known in the art is typically designed to achieve improved binding affinity to the target and / or reduced immunogenicity of the antibody at the receptor. In certain embodiments, humanized antibodies are modified to remove glycosylation sites to increase the affinity of the antibody to its cognate antigen. See, e.g., Co et al., MoI. Immunol, 30:1361-1367 (1993). In certain embodiments, humanized antibodies are produced using techniques such as "reshaping," "hyperchimerization," or "veneering / resurfacing." See, e.g., Vaswami et al., Annals of Allergy, Asthma, & Immunol. 81:105 (1998), Roguska et al., Prot. Engineer. 9:895-904 (1996), and U.S. Patent No. 6,072,035. In certain embodiments, such techniques typically reduce the immunogenicity of antibodies by reducing the number of foreign residues, but cannot prevent anti-idiotypic and anti-allotypic responses following repeated administration of the antibody.

[0150] In some cases, humanizing an antibody results in loss of antigen-binding ability. In certain embodiments, a humanized antibody is "backmutated." In certain embodiments, a humanized antibody is mutated to include one or more amino acid residues found in the donor antibody. See, e.g., Saldanha et al., MoI Immunol 36:709-19 (1999).

[0151] In certain embodiments, the complementarity determining regions (CDRs) of the light and heavy chain variable regions of an antibody against TrkA can be grafted into framework regions (FRs) of the same or another species. In certain embodiments, the CDRs of the light and heavy chain variable regions of an antibody against TrkA can be grafted into consensus human FRs. To create a consensus human FR, in certain embodiments, FRs from several human heavy or light chain amino acid sequences are aligned and a consensus amino acid sequence is identified. In certain embodiments, the FRs of an antibody against TrkA heavy or light chain are replaced with FRs of a different heavy or light chain. In certain embodiments, rare amino acids in the FRs of the heavy and light chains of an antibody against TrkA are not replaced and the remaining FR amino acids are replaced. A rare amino acid is a specific amino acid at a position that is not normally present in a FR. In certain embodiments, the grafted variable region from an antibody against TrkA can be used with a constant region that is different from the constant region of the antibody against TrkA. In certain embodiments, the grafted variable region is part of a single chain Fv antibody. CDR grafting is described, for example, in U.S. Pat. Nos. 6,180,370, 6,054,297, 5,693,762, 5,859,205, 5,693,761, 5,565,332, 5,585,089, and 5,530,101, as well as Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988); Winter, FEBS Letts., 430:92-94 (1998), which are incorporated by reference herein for any purpose.

[0152] As described herein, an antibody or antigen-binding fragment thereof that binds to TrkA may comprise a human (i.e., fully human) antibody and / or a portion thereof. In certain embodiments, nucleotide sequences encoding and amino acid sequences comprising heavy and light chain immunoglobulin molecules, particularly sequences corresponding to the variable regions, are provided. In certain embodiments, sequences corresponding to the complementarity determining regions (CDRs), particularly CDR1 through CDR3, are provided. According to certain embodiments, a hybridoma cell line expressing the immunoglobulin molecule is provided. According to certain embodiments, a hybridoma cell line expressing such a monoclonal antibody is provided. In certain embodiments, the hybridoma cell line is selected from at least one of the cell lines listed in Table 1, e.g., muPHD31, muPHD48, muPHD49, and / or muPHD50. In certain embodiments, a purified human monoclonal antibody against human TrkA is provided.

[0153] If mouse strains deficient in mouse antibody production are engineered with large fragments of the human Ig loci, it can be expected that the mice will produce human antibodies in the absence of mouse antibodies. The large human Ig fragments can maintain the diversity of large variable genes and the proper regulation of antibody production and expression. By taking advantage of mouse antibody diversification and selection mechanisms and the lack of immunological tolerance to human proteins, the human antibody repertoires recapitulated in these mouse strains can generate high affinity fully human antibodies against any antigen of interest, including human antigens. Using hybridoma technology, antigen-specific human MAbs with desired specificity can be generated and selected. Certain exemplary methods are described in WO 98 / 24893, U.S. Pat. No. 5,545,807, EP 546073, and EP 546073.

[0154] In certain embodiments, constant regions from species other than human can be used in conjunction with human variable regions.

[0155] The ability to clone and reconstruct megabase-sized human loci into yeast artificial chromosomes (YACs) and introduce them into the germline of mice provides an approach to elucidate the functional components of very large or loosely mapped loci as well as to generate useful models of human disease. Furthermore, utilizing such techniques to replace mouse loci with human ones may provide insight into the expression and regulation of human gene products during development, their communication with other systems, and their involvement in disease induction and progression.

[0156] Human antibodies avoid some of the problems associated with antibodies that have mouse or rat variable and / or constant regions. The presence of such mouse or rat derived proteins can result in rapid clearance of the antibody or an immune response by the patient against the antibody. To avoid the use of mouse or rat derived antibodies, fully human antibodies can be generated by introducing functional human antibody loci into a rodent, other mammal or animal, and causing the rodent, other mammal or animal to produce fully human antibodies.

[0157] A humanized antibody is an antibody that originally contained non-human antibody amino acid sequences, but in which at least some of these non-human antibody amino acid sequences have been replaced with human antibody sequences. This is in contrast to a human antibody, in which the antibody is (or can be) encoded by genes carried by a human.

[0158] Other antibodies provided are variants of the above-listed antibodies or antigen-binding fragments thereof formed by combinations or subportions of variable heavy and variable light chains shown in Table 1, each comprising a variable light chain and / or variable heavy chain having at least 50%, 50%-60%, 60%-70%, 70%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-97%, 97%-99%, or 99% or more identity to the amino acid sequence of the sequence in Table 1 (either the entire sequence or a subportion of the sequence, e.g., one or more CDRs). In some examples, such antibodies include at least one heavy chain and one light chain, while in other examples, the variants include two identical light chains and two identical heavy chains (or subportions thereof). In some embodiments, sequence comparison can be used to identify sections of the antibody that can be altered by observing mutations that affect binding and mutations that do not appear to affect binding. For example, by comparing similar sequences, it is possible to identify sections (such as specific amino acids) that can be altered and how they can be altered while maintaining (or improving) the functionality of the antibody or antigen-binding fragment thereof. In some embodiments, antibody variants include consensus groups and sequences between alternatives, as described above. The CDRs shown in Table 2 are defined according to the Kabat method (based on sequence variability, see, e.g., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication No. 91-3242, Kabat et al., (1991)).

[0159] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising a variable region comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from at least one of the sequences of SEQ ID NOs: 131, 133, 14, and 51. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising a variable region comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from at least one of the sequences of SEQ ID NOs: 131, 133, 14, and 51. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising a variable region comprising an amino acid sequence that is at least 99% identical to an amino acid sequence selected from at least one of the sequences of SEQ ID NOs: 131, 133, 14, and 51.

[0160] In some embodiments, the antibody or antigen-binding fragment thereof comprises a sequence that is at least 90%, 90-95%, and / or 95-99% identical to one or more CDRs from the CDRs in at least one of SEQ ID NOs: 131, 133, 14, and 51. In some embodiments, there are 1, 2, 3, 4, 5, or 6 CDRs (each at least 90%, 90-95%, and / or 95-99% identical to the above sequences).

[0161] In some embodiments, the antibody or antigen-binding fragment thereof comprises a sequence that is at least 90%, 90-95%, and / or 95-99% identical to one or more FRs from a FR in at least one of the sequences of SEQ ID NOs: 131, 133, 14, and 51. In some embodiments, there are 1, 2, 3, or 4 FRs (each at least 90%, 90-95%, and / or 95-99% identical to the above sequences).

[0162] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising a variable region comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from at least one of the sequences of SEQ ID NOs: 132, 134, 15, and 53. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising a variable region comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from at least one of the sequences of SEQ ID NOs: 132, 134, 15, and 53. In certain embodiments, the antigen-binding fragment thereof comprises a light chain comprising a variable region comprising an amino acid sequence that is at least 99% identical to an amino acid sequence selected from at least one of the sequences of SEQ ID NOs: 132, 134, 15, and 53.

[0163] In some embodiments, the antibody or antigen-binding fragment thereof comprises a sequence that is at least 90%, 90-95%, and / or 95-99% identical to one or more CDRs from the CDRs in at least one of SEQ ID NOs: 132, 134, 15, and 53. In some embodiments, there are 1, 2, 3, 4, 5, or 6 CDRs (each at least 90%, 90-95%, and / or 95-99% identical to the above sequences).

[0164] In some embodiments, the antibody or antigen-binding fragment thereof comprises a sequence that is at least 90%, 90-95%, and / or 95-99% identical to one or more FRs from a FR in at least one of the sequences of SEQ ID NOs: 132, 134, 15, and 53. In some embodiments, there are 1, 2, 3, or 4 FRs (each at least 90%, 90-95%, and / or 95-99% identical to the above sequences).

[0165] In view of the present disclosure, a skilled artisan will be able to use well-known techniques to determine suitable variants of the antibody or antigen-binding fragment thereof described herein. In certain embodiments, by targeting regions that are not believed to be important for activity, the skilled artisan can identify suitable regions of the molecule that can be altered without impairing activity. In certain embodiments, residues and portions of the molecule that are conserved between similar polypeptides can be identified. In certain embodiments, even regions that may be important for biological activity or structure can be subject to conservative amino acid substitutions without destroying biological activity or adversely affecting the polypeptide structure.

[0166] Additionally, one of skill in the art can review structure-function studies that identify residues in similar polypeptides that are important for activity or structure. In light of such comparisons, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues that are important for the activity or structure of the similar protein. One of skill in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues.

[0167] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to its structure in similar antibodies. Given such information, one skilled in the art can predict the arrangement of amino acid residues of an antibody in relation to the three-dimensional structure of the antibody. In certain embodiments, one skilled in the art can choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since said residues may be involved in important interactions with other molecules. Furthermore, one skilled in the art can generate test variants that contain a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known to one skilled in the art. The variants can be used to gather information about suitable variants. For example, if it is found that a change in a particular amino acid residue destroys, undesirably reduces, or produces inappropriate activity, variants with said change can be avoided. In other words, based on the information gathered from such routine experiments, one skilled in the art can easily determine which amino acids should be avoided for further substitution, either alone or in combination with other mutations.

[0168] In certain embodiments, the antigen-binding fragment variants include glycosylation variants in which the number and / or type of glycosylation sites are altered compared to the amino acid sequence of the parent polypeptide. In certain embodiments, the protein variants include a greater or lesser number of N-linked glycosylation sites than the native protein. The N-linked glycosylation sites are characterized by the sequence Asn-X-Ser or Asn-X-Thr, where the amino acid residue designated as X can be any amino acid residue except proline. Substitution of amino acid residues to create this sequence provides a potential new location for adding an N-linked carbohydrate chain. Alternatively, substitutions that remove this sequence remove an existing N-linked carbohydrate chain. Also provided is a rearrangement of the N-linked carbohydrate chain in which one or more N-linked glycosylation sites (usually naturally occurring) are removed and one or more new N-linked sites are generated. Additional preferred antibody variants include cysteine ​​variants in which one or more cysteine ​​residues are deleted or replaced with another amino acid (e.g., serine) compared to the parent amino acid sequence. Cysteine ​​variants are useful when antibodies need to be refolded into a biologically active conformation, such as after isolation of insoluble inclusion bodies. Cysteine ​​variants typically contain fewer cysteine ​​residues than the native protein, usually an even number to minimize interactions through unpaired cysteines.

[0169] According to certain embodiments, the amino acid substitutions (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) change the binding affinity for forming protein complexes, (4) change the binding affinity, and / or (4) confer or modify other physicochemical or functional properties to the polypeptide. According to certain embodiments, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) can be made in the native sequence (in certain embodiments, in the portion of the polypeptide outside the domains that form intermolecular contacts). In certain embodiments, conservative amino acid substitutions may not generally substantially alter the structural properties of the parent sequence (e.g., the substituted amino acid should not tend to disrupt helices occurring in the parent sequence or other types of secondary structures that characterize the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, ed., W.H. Freeman and Company, New York (1984)), Introduction to Protein Structure (C. Branden & J. Tooze, eds., Garland Publishing, New York, NY (1991)), and Thornton et al., Nature, 354:105 (1991), each of which is incorporated herein by reference.

[0170] In some embodiments, the variants are variants of the nucleic acid sequences of the antibodies or antigen-binding fragments thereof disclosed herein. Those skilled in the art will understand that the above description can be used to identify, evaluate, and / or create antibody and protein variants, as well as nucleic acid sequences that can encode those protein variants. Thus, nucleic acid sequences that encode those protein variants (as well as nucleic acid sequences that encode the antibodies or antigen-binding fragments thereof of Table 1, but that are different from those explicitly disclosed herein) are contemplated.

[0171] In certain embodiments, the antibody or antigen-binding fragment thereof is generated by immunization with an antigen (e.g., TrkA). In certain embodiments, the antibody can be produced by immunization with full-length TrkA, a soluble form of TrkA, the extracellular domain only, a splice variant form of TrkA, or a fragment thereof. In certain embodiments, the antibody of the present disclosure can be a polyclonal or monoclonal antibody and / or a recombinant antibody. In certain embodiments, the antibody of the present disclosure is a human antibody, prepared, for example, by immunization of a transgenic animal capable of producing human antibodies (see, e.g., PCT Application No. WO 93 / 12227).

[0172] In certain embodiments, certain strategies can be employed to engineer the intrinsic properties of an antibody, such as the affinity of the antibody for its target. Such strategies include, but are not limited to, using site-directed or random mutagenesis of a polynucleotide molecule encoding the antibody to generate antibody variants. In certain embodiments, such generation is followed by screening of antibody variants that exhibit the desired change, e.g., increased or decreased affinity.

[0173] In certain embodiments, the amino acid residues targeted in the mutagenesis strategy are amino acid residues in the CDRs. In certain embodiments, amino acids in the framework regions of the variable domain are targeted. In certain embodiments, such framework regions have been shown to contribute to the target binding properties of certain antibodies. See, e.g., Hudson, Curr. Opin. Biotech., 9:395-402 (1999) and references therein.

[0174] As will be appreciated, antibodies can also be expressed in cell lines other than hybridoma cell lines. Sequences encoding a particular antibody can be used to transform a suitable mammalian host cell. Transformation can be by any known method for introducing polynucleotides into a host cell, including, for example, packaging the polynucleotide into a virus (or viral vector) and transducing the host cell with the virus (or vector), or by transfection procedures known in the art, such as those exemplified in U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455, which are incorporated herein by reference. The transformation procedure used will vary depending on the host being transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus.

[0175] Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human epithelial kidney 293 cells, and many other cell lines. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with constitutive TrkA binding properties.

[0176] In certain embodiments, the antibody or antigen-binding fragment thereof comprises at least one immunoglobulin molecule of IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, and IgM isotypes. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a human kappa light chain and / or a human heavy chain. In certain embodiments, the heavy chain is an IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, or IgM isotype. In certain embodiments, the antibody or antigen-binding fragment thereof is cloned for expression in a mammalian cell. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a constant region other than any of the constant regions of the IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, and IgM isotypes.

[0177] Epitopes that anti-TrkA antibodies bind are provided. In some embodiments, epitopes that the antibodies disclosed herein bind are particularly useful. In some embodiments, antibodies that bind to any of the epitopes that the antibodies described herein bind, or antigen-binding fragments thereof, are useful. In some embodiments, epitopes that are bound by any of the antibodies listed in Table 1 are particularly useful. In some embodiments, the epitope is on the extracellular domain of TrkA.

[0178] In certain embodiments, a TrkA epitope can be utilized to prevent (e.g., reduce) binding of an anti-TrkA antibody, or antigen-binding fragment thereof, to TrkA. In certain embodiments, a TrkA epitope can be utilized to reduce binding of an anti-TrkA antibody, or antigen-binding fragment thereof, to TrkA. In certain embodiments, a TrkA epitope can be utilized to substantially inhibit binding of an anti-TrkA antibody, or antigen-binding fragment thereof, to TrkA.

[0179] In certain embodiments, TrkA epitopes can be used to isolate antibodies or antibodies or antigen-binding fragments thereof that bind to TrkA. In certain embodiments, TrkA epitopes can be used to generate antibodies or antigen-binding fragments thereof that bind to TrkA. In certain embodiments, TrkA epitopes or sequences that include TrkA epitopes can be used as immunogens to generate antibodies that bind to TrkA. In certain embodiments, TrkA epitopes can be administered to animals, and then antibodies that bind to TrkA can be obtained from the animals. In certain embodiments, TrkA epitopes or sequences that include TrkA epitopes can be used to disrupt normal TrkA-mediated activity, such as TrkA activation induced by NGF.

[0180] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein specifically bind to TrkA ECD. In some embodiments, the domain / region containing the residues that contact or are buried by the antibody can be identified by mutating specific residues in TrkA (e.g., wild-type antigen) and determining whether the antigen-binding fragment can bind to the mutant or mutated TrkA protein. By making multiple individual mutations, it is possible to identify residues that play a direct role in binding or that are in sufficient proximity to the antibody that the mutation may affect the binding between the antigen-binding fragment and the antigen. Knowledge of these amino acids can elucidate the domain or region of the antigen that contains the residues that contact the antigen-binding fragment or are covered by the antibody. The domain may include the binding epitope of the antigen-binding fragment. A specific example of this general approach utilizes an arginine / glutamic acid scanning protocol (see, for example, Nanevicz, T., et al., 1995, J. Biol. Chem., 270:37, 21619-21625 and Zupnick, A., et al., 2006, J. Biol. Chem., 28_L:29, 20464-20473). In general, arginine and glutamic acid are substituted (usually individually) for amino acids in the wild-type polypeptide because these amino acids are charged and bulky and may disrupt binding between the antigen-binding fragment and the antigen in the region of the antigen where the mutation has been introduced. Arginines present in the wild-type antigen are replaced with glutamic acid. A variety of such individual mutants are obtained and the collected binding results are analyzed to determine which residues affect binding.

[0181] As mentioned above, the residues directly involved in binding or covered by the antigen-binding fragment thereof can be identified from the scan results or Cryo-EM. Thus, these residues can provide an indication of the domain or region of SEQ ID NO: 119 (or SEQ ID NO: 120) that contains the binding region to which the antibody or antigen-binding fragment of the present invention binds. As can be seen from the results summarized in Example 8, in some embodiments, the antigen-binding fragment thereof binds to a domain that includes at least one of amino acids Q176, H178, G179, Q180, and P187 of SEQ ID NO: 119.

[0182] In the present application, the antibody or antigen-binding fragment thereof can recognize an epitope of the TrkA extracellular domain (ECD) that includes amino acid residues Q176, H178, G179, Q180, and / or P187 of SEQ ID NO: 119. For example, the epitope can include one or more (e.g., 2, 3, 4, or 5) amino acid residues Q176, H178, G179, Q180, and P187 of SEQ ID NO: 119.

[0183] In some cases, the antibody or antigen-binding fragment thereof may be capable of specifically binding to Q176, H178, G179, Q180 and / or P187 of SEQ ID NO: 119. For example, the antibody or antigen-binding fragment thereof may specifically bind to one or more (e.g., 2, 3, 4, or 5) amino acid residues of Q176, H178, G179, Q180, and P187 of SEQ ID NO: 119.

[0184] In some cases, the ECD of TrkA comprises the amino acid sequence set forth in SEQ ID NO: 120. In the present application, TrkA can comprise the amino acid sequence set forth in SEQ ID NO:119.

[0185] In some cases, the distance between an antigen atom and the corresponding interaction site of the antibody or antigen-binding fragment may be about 4.00 Å or less, for example, 3.76 Å, 3.62 Å, 3.43 Å, 3.36 Å, 3.13 Å, 3.06 Å, and / or 2.68 Å.

[0186] In some cases, the interaction site of an antibody that binds to antigen atom Q176 may be heavy chain W33 of SEQ ID NO: 86 or a corresponding residue (e.g., the correspondence can be determined by sequence alignment). In some cases, the interaction site of an antibody that binds to antigen atom H178 may be light chain Y90 of SEQ ID NO: 91 or a corresponding residue. In some cases, the interaction site of an antibody that binds to antigen atom G179 may be heavy chain H35 of SEQ ID NO: 86 or a corresponding residue. In some cases, the interaction site of an antibody that binds to antigen atom G179 may be light chain Y90 of SEQ ID NO: 91 or a corresponding residue. In some cases, the interaction site of an antibody that binds to antigen atom Q180 may be heavy chain W104 of SEQ ID NO: 86 or a corresponding residue. In some cases, the interaction site of an antibody that binds to antigen atom Q180 may be light chain Y90 of SEQ ID NO: 91 or a corresponding residue. In some cases, the interaction site of an antibody that binds to antigen atom P187 may be light chain Y31 of SEQ ID NO: 91 or a corresponding residue.

[0187] In another aspect, the present application provides a method for screening or obtaining a TrkA antibody that does not substantially inhibit the binding of TrkA to NGF. The method may include using an epitope of the TrkA extracellular domain (ECD) that includes amino acid residues Q176, H178, G179, Q180, and / or P187 of SEQ ID NO: 119. For example, the epitope of the TrkA extracellular domain (ECD) may include one or more (e.g., 2, 3, 4, or 5) amino acid residues Q176, H178, G179, Q180, and P187 of SEQ ID NO: 119. For example, the TrkA antibody may have one or more of the following properties: a TrkA antibody that is about 5*10 as measured by Octet or SPR; -8 K under M Dthe antibody or antigen-binding fragment thereof may specifically recognize or bind to an epitope of the TrkA extracellular domain (ECD), and the epitope may include amino acid residues Q176, H178, G179, Q180, and / or P187 of SEQ ID NO: 119.

[0188] In some embodiments, a truncated or truncated TrkA protein (e.g., a human TrkA protein) or its ECD may be used to immunize an animal (e.g., a mouse, rabbit, or other non-human animal) or introduced into an antibody-producing cell (e.g., a B cell, e.g., a human B cell) and antibodies that specifically bind to the truncated TrkA protein may be further analyzed or selected. A truncated TrkA protein includes amino acid residues Q176, H178, G179, Q180, and / or P187 of SEQ ID NO:119.

[0189] In some cases, a mutated or modified TrkA protein may be used in which one or more residues corresponding to Q176, H178, G179, Q180, and / or P187 of SEQ ID NO: 119 have been mutated to a different residue or deleted. If a candidate antibody or antigen-binding fragment thereof does not bind to the mutated or modified TrkA protein or binds with significantly lower affinity (e.g., K D If the value is 20% higher, 25% higher, 30% higher, 35% higher, 40% higher, 45% higher, 50% higher, 55% higher, 60% higher, 65% higher, 70% or more higher), the antibody or antigen-binding fragment thereof may be further analyzed or selected as it may be considered an antibody (or antigen-binding fragment thereof) having a desired property (e.g., a property contained in an antibody or antigen-binding fragment of the present disclosure).

[0190] In another aspect, the present application provides the use of an epitope of the TrkA extracellular domain (ECD) for obtaining or screening a TrkA antibody that does not substantially inhibit the binding of TrkA to NGF, said epitope comprising amino acid residues Q176, H178, G179, Q180, and / or P187 of SEQ ID NO: 119.

[0191] In another aspect, the disclosure provides the use of an epitope of the TrkA extracellular domain (ECD) in the manufacture of an agent for obtaining or screening a TrkA antibody that does not substantially inhibit binding of TrkA to NGF, wherein the epitope comprises amino acid residues Q176, H178, G179, Q180, and / or P187 of SEQ ID NO: 119.

[0192] In some cases, the antibody or antigen-binding fragment thereof may compete with a reference antibody for binding to TrkA, the reference antibody comprising light chain CDR1-3 and heavy chain CDR1-3, wherein light chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 96, 97, and 98, respectively, and heavy chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 93, 94, and 95, respectively.

[0193] In some cases, the reference antibody comprises light chain CDR1-3 and heavy chain CDR1-3, wherein light chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 117, 118, and 41, respectively, and heavy chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NO: 114 (SX 1 WX 2 Q, here X 1 is H, Y, X 2 includes the amino acid sequences set forth in I or M), 115, and 116.

[0194] In some cases, the reference antibody comprises light chain CDR1-3 and heavy chain CDR1-3, where light chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 13, respectively, and heavy chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 1, 4, and 7, respectively.

[0195] In some cases, the reference antibody comprises light chain CDR1-3 and heavy chain CDR1-3, where light chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 28, 30, and 32, respectively, and heavy chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 23, and 26, respectively.

[0196] In some cases, the reference antibody comprises light chain CDR1-3 and heavy chain CDR1-3, where light chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 56, 58, and 60, respectively, and heavy chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 62, 65, and 68, respectively.

[0197] In some cases, the reference antibody comprises light chain CDR1-3 and heavy chain CDR1-3, where light chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 56, 58, and 60, respectively, and heavy chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 73, and 68, respectively.

[0198] In some cases, the reference antibody comprises light chain CDR1-3 and heavy chain CDR1-3, where light chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 56, 58, and 60, respectively, and heavy chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 75, and 68, respectively.

[0199] In some cases, the reference antibody comprises light chain CDR1-3 and heavy chain CDR1-3, where light chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 56, 77, and 60, respectively, and heavy chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 62, 65, and 68, respectively.

[0200] In some cases, the reference antibody comprises light chain CDR1-3 and heavy chain CDR1-3, where light chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 56, 77, and 60, respectively, and heavy chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 73, and 68, respectively.

[0201] In some cases, the reference antibody comprises light chain CDR1-3 and heavy chain CDR1-3, where light chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 56, 77, and 60, respectively, and heavy chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 75, and 68, respectively.

[0202] In some cases, the reference antibody comprises light chain CDR1-3 and heavy chain CDR1-3, where light chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 80, 82, and 32, respectively, and heavy chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 62, 79, and 26, respectively.

[0203] In some cases, the reference antibody comprises light chain CDR1-3 and heavy chain CDR1-3, where light chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 30, and 32, respectively, and heavy chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 87, and 26, respectively.

[0204] In some cases, the reference antibody comprises light chain CDR1-3 and heavy chain CDR1-3, where light chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 28, 39, and 41, respectively, and heavy chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 37, respectively.

[0205] In some cases, the reference antibody comprises light chain CDR1-3 and heavy chain CDR1-3, where light chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 103, and 41, respectively, and heavy chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 101, 102, and 37, respectively.

[0206] In some cases, the reference antibody comprises light chain CDR1-3 and heavy chain CDR1-3, where light chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 103, and 41, respectively, and heavy chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 109, 110, and 111, respectively.

[0207] In some cases, the reference antibody comprises light chain CDR1-3 and heavy chain CDR1-3, where light chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 39, and 41, respectively, and heavy chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 37, respectively.

[0208] In some cases, the reference antibody comprises light chain CDR1-3 and heavy chain CDR1-3, where light chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 39, 108, and 41, respectively, and heavy chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 109, 110, and 111, respectively.

[0209] In some cases, the reference antibody comprises light chain CDR1-3 and heavy chain CDR1-3, where light chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 50, 39, and 41, respectively, and heavy chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 47, 48, and 49, respectively.

[0210] In some embodiments, the antibody or antigen-binding fragment thereof comprises at least one of light chain CDRs 1 to 3 of a light chain variable region, wherein the light chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 132, 134, 15, and 53.

[0211] In some embodiments, the antibody or antigen-binding fragment thereof comprises at least one of heavy chain CDRs 1 to 3 of a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 131, 133, 14, and 51.

[0212] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain CDR1, wherein the light chain CDR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 96, 117, 9, and 50.

[0213] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain CDR1, wherein the light chain CDR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 9, 28, 50, 56, 80, and 88.

[0214] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain CDR2, wherein the light chain CDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 97, 118, and 11.

[0215] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain CDR2, wherein the light chain CDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 11, 30, 39, 58, 77, 82, 103, and 108.

[0216] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain CDR3, wherein the light chain CDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 98, 13, and 41.

[0217] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain CDR3, wherein the light chain CDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 13, 32, 41, 60.

[0218] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region, wherein the light chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 132, 134, 15, and 53.

[0219] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain CDR1, wherein the heavy chain CDR1 is SEQ ID NO: 93, 114 (SX 1 WX 2 Q, here X 1 is H or Y, and X 2 is I or M), 1, and 47.

[0220] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain CDR1, wherein the heavy chain CDR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1, 20, 45, 47, 62, 101, and 109.

[0221] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain CDR2, wherein the heavy chain CDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 94, 115, 4, and 48.

[0222] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain CDR2, wherein the heavy chain CDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 4, 23, 46, 48, 65, 73, 75, 87, 102, and 110.

[0223] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain CDR3, wherein the heavy chain CDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 95, 116, 7, and 49.

[0224] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain CDR3, wherein the heavy chain CDR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 7, 26, 37, 49, 68, and 111.

[0225] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 131, 133, 14, and 51.

[0226] In some embodiments, the antibody or antigen-binding fragment thereof comprises: 1) light chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 96, 97, and 98, respectively, and heavy chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 93, 94, and 95, respectively; 2) light chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 117, 118, and 41, respectively, and heavy chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 114 (SX 1 WX 2 Q, where X 1 H or Y, and X 21) heavy chain CDR1 to 3 comprising the amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 4) light chain CDR1 to 3 comprising the amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 1010, 109, 1011 5) heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 62, 65, and 68, respectively; 6) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 56, 58, and 60, respectively, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 20, 73, and 68, respectively; 7) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 56, 58, and 60, respectively, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 20, 75, and 68, respectively; 8) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 56, 77, and 60, respectively. 9) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 56, 77, and 60, respectively, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 20, 73, and 68, respectively; 10) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 56, 77, and 60, respectively, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 20, 75, and 68, respectively; 11) SEQ ID NO: 80, 82, and 32, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 62, 79, and 26, respectively; 12) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 88, 30, and 32, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 20, 87, and 26, respectively; 13) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 28, 39, and 41, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 37, respectively;14) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 88, 103, and 41, respectively, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 101, 102, and 37, respectively; 15) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 88, 103, and 41, respectively, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 109, 110, and 111, respectively; 16) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 88, 39, and 41, respectively, and and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 37, respectively; 17) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 39, 108, and 41, respectively, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 109, 110, and 111, respectively; or 18) light chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 50, 39, and 41, respectively, and heavy chain CDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 47, 48, and 49, respectively.

[0227] In some embodiments, the antibody or antigen-binding fragment thereof comprises: 1) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 132, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 131; 2) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 134, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 133; 3) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 15, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 14; 4) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 18, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16; 5) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 90, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 89; 6) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 91, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 86; 7) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 91, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 92; 8) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 61, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70; 10) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 61, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 74; 11) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 78, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70; 12) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 78, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 74; 13) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 78, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 76; 14) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 85, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 84; 15) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 44, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 42; 16) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 100, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 99; 17) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 104;and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 105, 18) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 104, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 112, 19) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 107, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 106, 20) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 113, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 112, or 21) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 53, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 51.

[0228] Antibodies or antigen-binding fragments may also include homologs or variants thereof having substantially the same function / property. In some cases, a homolog or variant may have an amino acid sequence that differs from the amino acid sequence of an antibody or antigen-binding fragment of the present disclosure by at least one amino acid. For example, a homolog or variant may be a polypeptide that differs from an antibody or antigen-binding fragment thereof by the addition, deletion, or substitution of one or more amino acids, such as 1-50, 1-40, 1-30, 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acids. In some cases, a homolog or variant may be a polypeptide that has at least 80% sequence identity to an antibody or antigen-binding fragment thereof. For example, a homologue or variant may be a polypeptide having 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) sequence identity to an antibody or antigen-binding fragment thereof.

[0229] The term "percent (%) sequence identity" as used in the context of the polypeptide sequences identified herein generally refers to the percentage of amino acid residues or nucleotides in a query sequence that are identical to the amino acid residues or nucleotides of a second reference polypeptide sequence or a portion thereof, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment to determine percent identity of amino acid / nucleotide sequences can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. Percent identity may be measured over the entire length of the defined polypeptide / polynucleotide sequence, or over a shorter length, for example, the length of a fragment taken from a larger defined polypeptide / polynucleotide sequence. It is understood that any fragment length supported by a sequence shown in this specification, a table, a figure, or the sequence listing can be used to describe the length over which the percentage of identity can be determined.

[0230] In some cases, one or more (e.g., 2, 3, 4, or 5) of the following residues or corresponding residues are not altered or mutated in a variant or homologue of an antibody or antigen-binding fragment thereof compared to its parent sequence: heavy chain W33 of SEQ ID NO: 86 or a corresponding residue (e.g., correspondence can be determined by sequence alignment), light chain Y90 of SEQ ID NO: 91 or a corresponding residue, heavy chain H35 of SEQ ID NO: 86 or a corresponding residue, heavy chain W104 of SEQ ID NO: 86 or a corresponding residue, and / or light chain Y31 of SEQ ID NO: 91 or a corresponding residue.

[0231] In the present application, the CDR sequences may be classified according to a specific CDR classification standard (e.g., Kabat method). It should be noted that if the amino acid sequence of the TrkA antibody or its antigen-binding fragment determined according to a specific CDR classification standard (e.g., Kabat method) is the same as the CDR sequence defined in the present application, the TrkA antibody or its antigen-binding fragment also falls within the scope of protection of the present application. According to different CDR classification standards, the exact identification of the CDR position may be slightly different, therefore, the present application includes not only the CDRs shown in the sequence listing, but also the CDRs contained in the VH and VL domains using other classification methods such as Chothia, extended Chothia, or IMGT.

[0232] In another aspect, the application provides a fusion protein comprising an antibody or antigen-binding fragment thereof of the present disclosure.

[0233] In another aspect, the application provides a protein complex (such as an immunoconjugate) comprising an antibody or antigen-binding fragment thereof of the present disclosure.

[0234] Nucleic acids, vectors, cells and preparation methods In another aspect, the disclosure provides an isolated nucleic acid or molecule encoding an antibody or antigen-binding fragment thereof, or a fusion protein.

[0235] The isolated nucleic acid comprises one or more nucleic acid molecules, each encoding at least a portion of the antibody or antigen-binding fragment of the present disclosure.For example, the isolated nucleic acid comprises at least two nucleic acid molecules, one of which encodes an antibody heavy chain or fragment thereof, and the other of which encodes an antibody light chain or fragment thereof.In some cases, the isolated nucleic acid may encode a fusion protein.

[0236] The isolated nucleic acid or isolated nucleic acids can be synthesized using recombinant techniques well known in the art. For example, the isolated nucleic acid or isolated nucleic acids can be synthesized using an automated DNA synthesizer. Standard recombinant DNA and molecular cloning techniques include those described in Sambrook, J., Fritsch, EF, Maniatis, T. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989) (Maniatis) and TJ Silhavy, ML Bennan, LW Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and Ausubel, FM et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987). Briefly, the nucleic acid of the present invention can be prepared from genomic DNA fragments, cDNA, and RNA, all of which can be directly extracted from cells or recombinantly produced by various amplification processes, including but not limited to PCR and RT-PCR.

[0237] In direct chemical synthesis of nucleic acids, 3'-blocked and 5'-blocked nucleotide monomers are typically added sequentially to the terminal 5'-hydroxyl group of a growing nucleotide polymer chain. Each addition is effected by nucleophilic attack at the 3' position of the added monomer by the terminal 5'-hydroxyl group of the growing chain. The added monomers are typically phosphorus derivatives, such as phosphotriesters, phosphoramidites, etc. See, e.g., Matteuci et al., Tet. Lett. 521:719 (1980), and also U.S. Patent No. 4,500,707 to Caruthers et al., and U.S. Patent Nos. 5,436,327 and 5,700,637 to Southern et al.

[0238] In another aspect, the disclosure provides a vector comprising the isolated nucleic acid molecule.

[0239] The vector can be any linear nucleic acid, a plasmid, a phagemid, a cosmid, an RNA vector, a viral vector, etc. Non-limiting examples of viral vectors include retroviruses, adenoviruses, adeno-associated viruses, etc. In some cases, the vector is an expression vector, e.g., a phage display vector.

[0240] An expression vector may be suitable for certain types of host cells but not for other types of host cells. For example, an expression vector can be introduced into a host organism and the viability and expression of the gene / polynucleotide contained in the vector can be monitored.

[0241] Expression vectors may also contain one or more selectable marker genes whose expression confers one or more phenotypic traits useful for selecting or identifying host cells harboring the expression vector. Non-limiting examples of suitable selectable markers for eukaryotic cells include dihydrofolate reductase and neomycin resistance.

[0242] The subject vectors can be stably or transiently introduced into the host cells by various established techniques. For example, one method involves calcium chloride treatment, where the expression vector is introduced via calcium precipitate. Other salts, such as calcium phosphate, can be used following a similar procedure. In addition, electroporation (i.e., applying an electric current to increase the permeability of cells to nucleic acids) can also be used. Other examples of transformation methods include microinjection, DEAE-dextran-mediated transformation, and heat shock in the presence of lithium acetate. Lipid complexes, liposomes, and dendrimers can also be used to transfect host cells.

[0243] In another aspect, the disclosure provides a cell (eg, an isolated cell such as a host cell) comprising an isolated nucleic acid molecule of the disclosure or a vector of the disclosure.

[0244] The cell may express the antibody of the present invention, or an antigen-binding fragment thereof, or a fusion protein of the present invention. The cell may be a eukaryotic or prokaryotic cell. Suitable cells may be transformed or transfected with the nucleic acid or vector of the present invention and utilized for the expression and / or secretion of the antibody, antigen-binding fragment thereof, or fusion protein. For example, the cell may be an E. coli cell, other bacterial host cell, yeast cell, or various higher eukaryotic cells.

[0245] In another aspect, the invention provides a method for producing an antibody or antigen-binding fragment thereof, or a fusion protein of the invention, said method comprising culturing a cell of the invention under conditions allowing expression of the antibody, antigen-binding fragment thereof, or fusion protein.

[0246] The method may optionally further comprise harvesting the antibody or antigen-binding fragment thereof, or the fusion protein of the disclosure.

[0247] composition In another aspect, the disclosure provides a composition comprising an antibody or antigen-binding fragment thereof, a fusion protein, a protein complex, an isolated nucleic acid molecule, a vector, and / or a cell of the disclosure, and optionally a pharma- ceutically acceptable excipient.

[0248] Optionally, the pharma- ceutically acceptable excipient may include a buffering agent. Optionally, the pharma- ceutically acceptable excipient may include an amino acid.

[0249] In some embodiments, the pH of the composition may be between 1 and 13, for example, the pH may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13.

[0250] In some cases, the composition may further comprise an effective amount of an additional therapeutically active ingredient, for example, an additional therapeutically active ingredient for treating a disease or disorder associated with inappropriate expression or function of TrkA. Each active ingredient may be present in the pharmaceutical composition in a medicamentously active amount. In the composition, the antibody, fragment thereof, of the present application may or may not be conjugated to an additional active ingredient.

[0251] The following describes non-limiting exemplary pharmaceutical compositions and their preparation methods. The pharmaceutical composition may be in a form suitable for oral administration, such as tablets, capsules, pills, powders, sustained release formulations, solutions, suspensions, suitable for parenteral injection, such as sterile solutions, suspensions or emulsions, suitable for topical administration, such as ointments or creams, or suitable for rectal administration, such as suppositories. The pharmaceutical composition may be in a unit dosage form suitable for single administration of an accurate dosage. In some cases, the pharmaceutical composition may be a liquid pharmaceutical composition.

[0252] The pharmaceutical compositions of the present disclosure can be provided as individual dosage forms, each of which contains a predetermined amount of active ingredient as powder or granules, solution, or suspension in aqueous or non-aqueous liquid. Such dosage forms can be prepared by any method known to those skilled in the art, and can include, for example, combining active ingredient with a carrier that constitutes one or more other ingredients. In general, the composition is prepared by uniformly and intimately mixing active ingredient with liquid carrier or finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired shape.

[0253] The antibodies, antigen-binding fragments thereof, or fusion proteins of the invention can be combined in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier can take a variety of forms depending on the form of preparation desired for administration.

[0254] The composition may further include one or more pharma- ceutically acceptable additives and excipients, including, but not limited to, anti-adherents, anti-foaming agents, buffers, polymers, antioxidants, preservatives, chelating agents, viscosity modifiers, tonicity agents, flavoring agents, coloring agents, odorants, opacifiers, suspending agents, binders, fillers, plasticizers, lubricants, and / or mixtures thereof.

[0255] The pharmaceutical composition of the present disclosure may comprise a therapeutically effective amount of an active agent (e.g., an antibody, an antigen-binding fragment thereof, or a fusion protein of the present disclosure). A therapeutically effective amount is an amount of the subject pharmaceutical composition that can (at least partially) prevent and / or cure a condition or disorder (e.g., chronic pain) and / or its complications in a subject suffering from or at risk of developing said condition or disorder. The specific amount / concentration of the active agent included will vary depending on the method of administration and the needs of the patient, and can be determined, for example, based on the patient's volume, viscosity, and / or weight, etc. For example, a suitable dosage may be about 0.1 mg or 1 mg / kg / day to about 50 mg / kg / day, although in some cases the dosage may be even higher. It should be understood that these specific dosages can be conveniently adjusted by a person skilled in the art (e.g., a physician or pharmacist) based on the particular patient's condition, formulation, and / or disease.

[0256] Medical Uses and Treatments In another aspect, the invention provides the use of an antibody or antigen-binding fragment thereof, fusion protein, protein complex, isolated nucleic acid molecule, vector, and / or cell of the invention in the manufacture of a medicament for the prevention and / or treatment of a disease or disorder associated with inappropriate expression or function of TrkA.

[0257] In a further aspect, the application provides a method of preventing and / or treating a disease or disorder in a subject in need thereof comprising administering to the subject an effective amount of an antibody or antigen-binding fragment, fusion protein, protein complex, isolated nucleic acid molecule, vector, cell, and / or composition of the present disclosure, wherein the disease or disorder is a disease or disorder associated with inappropriate expression or function of TrkA.

[0258] For example, the disease or disorder may include pain.

[0259] For example, the pain may include chronic pain.

[0260] For example, the disease or disorder may include chronic pain of nociceptive, non-nociceptive, traumatic, inflammatory, neuropathic, proliferative, or mixed etiology. For example, the disease or disorder may include chronic pain resulting from musculoskeletal or neuropathic disorders.

[0261] For example, the disease or disorder may include post-operative pain, rheumatoid arthritis pain, neuropathic pain, and / or osteoarthritis pain.

[0262] In other embodiments, the pain is acute or chronic pain caused by musculoskeletal or neuropathic disorders.Specific non-limiting examples of pain that can be prevented and / or treated by the present application include, for example, postoperative pain, rheumatoid arthritis pain, neuropathic pain (including radicular pain CLBP, DNP, and LSR), and osteoarthritis pain (including non-radicular pain).In some cases, the pain is chronic pain caused by both musculoskeletal and neuropathic disorders.In other embodiments, the pain is visceral pain (such as, for example, chronic prostatitis, interstitial cystitis, or chronic pelvic pain).

[0263] For example, the disease or disorder may be pain associated with any of the following, including, but not limited to, pancreatitis, kidney stones, endometriosis, IBD, Crohn's disease, post-operative adhesions, gallbladder stones, headaches, dysmenorrhea, musculoskeletal pain, sprains, visceral pain, ovarian cysts, prostatitis, cystitis, interstitial cystitis, post-operative pain, migraine, trigeminal neuralgia, burn and / or wound pain, traumatic pain, neuropathic pain, musculoskeletal pain, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, peri-articular lesions, tumor pain, bone metastasis pain, and / or HIV infection pain.

[0264] In another aspect, the disclosure provides the use of an antibody or antigen-binding fragment thereof, a fusion protein, or a protein complex in the manufacture of a medicament for determining the presence and / or amount of TrkA in a sample.

[0265] In another aspect, the present disclosure provides a method for determining the presence and / or amount of TrkA in a sample, the method comprising: a) contacting the sample with an antibody or antigen-binding fragment, fusion protein, or protein complex of the disclosure; and b) determining the presence and / or amount of the antibody or antigen-binding fragment, fusion protein, or protein complex bound to the sample. EXAMPLES

[0266] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present application, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to be exhaustive or the only experiments performed. Efforts have been made to ensure accuracy in numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviations exist.

[0267] Example 1. Generation of anti-TrkA monoclonal antibodies TrkA recombinant protein for immunology, binding and functional assays The extracellular domain (ECD) of human TrkA protein (SEQ ID NO: 120) was genetically synthesized and subcloned into a pcDNA3.4-based expression vector fused to a His tag at the C-terminus and a signal peptide and flag tag (PHA) at the N-terminus. The resulting plasmid was transiently transfected into 293-F cells and incubated in a CO 2 -incubator equipped with a rotary shaker. 2 The cells were cultured in an incubator for 5–7 days. The supernatant containing the recombinant protein was collected and clarified by centrifugation, and the protein was then purified by one-step immobilized metal affinity chromatography. The purified protein was buffer-exchanged into phosphate-buffered saline (PBS) and stored in aliquots at -80°C in a freezer.

[0268] Immunization, hybridoma fusion and cloning One group of BALB / c mice (5 mice / group) was immunized with the above purified proteins according to the schedule in Table 3. TrkA ECD protein was mixed 1:1 with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) to obtain a stable oil-in-water emulsion. CFA was used only in the first immunization. Subsequent immunizations were performed with PBS containing IFA. The injection dose was 25–50 μg / 200 L per mouse. After immunization, serum samples were collected and antibody responses (titers) against the immunizing antigens were measured by indirect ELISA and FACS (see below). Four days after the boost, spleen cells were isolated and fused with SP2 / 0 myeloma cells by electrical cell fusion to induce cell fusion and form hybridomas. The fused cells were seeded into 96-well plates, and 50 plates were used for each fusion.

[0269] [Table 3] Determine the binding activity of TrkA antibodies Hybridoma supernatants were first screened for human TrkA by indirect ELISA. Briefly, purified PHA was diluted in PBS buffer to a final concentration of 1 μg / mL. 100 μL of diluted antigen was added to each well of a 96-well plate and incubated overnight at 4 °C. The plate was washed three times with PBST (PBS containing 0.05% Tween-20, pH 7.4) and then blocked by adding 200 μL / well of 2% bovine serum albumin (BSA) diluted in PBST and incubating at room temperature for 2 h. The blocking solution was removed and the plate was washed three times with 300 μL of PBST. Dilution solutions of primary and secondary antibodies, and substrate solution were prepared. 100 μL of diluted primary antibody was pipetted into each well and incubated at room temperature for 1 h. The well contents were removed and washed three times with 300 μL of PBST buffer. PBS was removed from the plate. 100 μL of diluted Peroxidase-AffiniPure Goat Anti-Mouse IgG was added to each well and incubated at 37°C for 1 hour. The contents of the wells were removed and the wells were washed three times with 300 μL of PBST. 100 μL of TMB substrate solution was added to the wells. After sufficient color development, 100 μL of stop solution was added to the wells to stop the reaction. The absorbance (OD:450) of each well was read with a microplate reader and the data was analyzed.

[0270] ELISA-positive antibody-producing clones were further validated by fluorescence-activated cell sorting (FACS) using conventional methods. Briefly, CellSensor® TrkA-NFAT-bla CHO-K1 cells (cells overexpressing human TrkA) were stained with anti-TrkA antibody in U-bottom 96-well plates. Cells were diluted in ice-cold PBS at 2 × 10 6The cells were resuspended in 100 μL of ice-cold PBS. Dilutions of primary and secondary antibodies were prepared, and 100 μL of diluted primary antibodies were added to each well and incubated for 1 h at 4 °C in the dark. The cells were washed twice by centrifugation at 2500 rpm for 3 min and resuspended in ice-cold PBS. APC goat anti-mouse IgG was diluted to 1 μg / mL in cold PBS, and the cells were then resuspended in 100 μL of this solution. The cells were washed twice by centrifugation at 2500 rpm for 3 min and resuspended in 100 μL of ice-cold PBS. The cell suspension was immediately stored at 4 °C in the dark. The cells were analyzed on a flow cytometer as soon as possible.

[0271] Subcloning Hybridomas that showed positive binding in both ELISA and FACS assays were then tested in functional assays to identify antibodies with the desired functional activity. Antibodies with positive functional activity were further subcloned by limiting dilution and characterized by FACS and functional assays. Subclones selected through functional assays were defined as monoclonal antibodies. Selected subclones were cultured in Hybridoma-SFM medium.

[0272] Monoclonal antibody expression and purification Positive hybridomas were first cultured in Hybridoma-SFM medium, and then the cells were incubated in Hybridoma-SFM medium at 5% CO 2 The cells were cultured in an incubator at 37°C for 4 days. After the culture, cell viability and protein production were evaluated.

[0273] The heavy and light chain variable region sequences of the antibody to be expressed were inserted into pCDNA3.4-hIgG4 or pCDNA3.4-hKappa vectors, respectively. The resulting vector plasmids were extracted and verified by sequencing. The verified plasmids were transfected into human 293F cells containing PEI and continuously cultured. 293F cells were cultured to logarithmic growth phase in serum-free medium (Shanghai OPM Bioscience, OPM-293CD03) for cell transfection. 3 μg of antibody light chain plasmid and 2 μg of antibody heavy chain plasmid were dissolved in 1 mL of Opti-MEM® I reduced serum medium (GIBCO, 31985-070), mixed well, added 20 μg of PEI, mixed well, incubated at room temperature for 15 min, and added to 5 mL of cells. The cell culture conditions were as follows: 5% CO 2 , 37°C, 125 rpm / min. Feeder medium was replenished on day 1. Cells were cultured for an additional 4 days.

[0274] All cells were collected and centrifuged at 4000 rpm, 4°C for 30 min. The supernatant containing the antibodies was collected. Antibody purification was performed using GenScript Protein A MagBeads according to the manufacturer's instructions. Briefly, 1) Binding. The clarified supernatant containing the antibody of interest was incubated with MagBeads for 2 h at room temperature. 2) Washing. The beads were collected using a magnetic separation rack and the supernatant was discarded. 1 mL of PBS buffer was added to the tube and mixed well, the beads were collected using a magnetic separation rack and the supernatant was discarded. The washing step was repeated three more times. 3) Elution. 100 μL of elution buffer (0.1 M glycine, pH 3.0) was added to the tube and mixed well. Incubated for 5 min at room temperature with occasional mixing. The beads were collected using a magnetic separation rack and the supernatant containing the eluted IgG was transferred to a clean tube. To neutralize the pH, 10 μL of neutralization buffer (1 M Tris, pH 8.5) was added to each 100 μL of elution. Protein concentration was determined by measuring absorbance at 280 nm, and the purified antibodies were aliquoted and stored in a freezer at -80°C. Three control antibodies, anti-TNP (with VH and VL shown in SEQ ID NOs: 125 and 126, respectively), tanezumab (with VH and VL shown in SEQ ID NOs: 127 and 128, respectively), and fasinumab (with VH and VL shown in SEQ ID NOs: 129 and 130, respectively), were also prepared by the above method.

[0275] Antagonistic effects on TrkA activation using NFAT reporter assay CellSensor® TrkA-NFAT-bla CHO-K1 cells (K1516, Life Technologies) contain a beta-lactamase reporter gene under the control of an NFAT response element, which has been stably integrated into CHO-K1 cells, which also stably express the human TrkA gene. Thus, when cells are stimulated with nerve growth factor 2.5s (NGF2.5s), TrkA signaling is activated and can be detected with a special fluorescent substrate for beta-lactamase. Using this cell line, samples can be tested for antagonistic effects on the NGF-TrkA signaling pathway. Cellsensor TrkA-NFAT-bla CHO-K1 cells (K1516, Life Technologies) were seeded in 384-well plates with 32 μL of medium the day before the experiment, according to the Invitrogen cellsensor_TrkA NFAT bla_CHOK1_manual. Cells were cultured at 37°C / 5% CO 2 The antagonist assay plates were pre-incubated with 3-fold serial dilutions of antibodies for 30 minutes in a humidified 37°C / 5% CO incubator. Then, 4 μL of an 11X EC80 stock solution of NGF was added. The antagonist assay plates were then incubated at 37°C / 5% CO in a humidified 37°C / 5% CO incubator. 2 Incubated in an incubator for 5 hours. 6X LiveBLAzer TM -FRET B / G substrate (CCF4-AM) mixture was prepared according to the manufacturer's instructions. 8 μL of 6X substrate mixture was added to each well. The plate was covered to protect from light and evaporation and incubated for 2 h at room temperature. Cell-free wells were used as background. FRET signals (405 nm excitation, and 460 nm and 530 nm emission) were acquired from a microplate reader (C5, Biotek) and background signals were subtracted. The ratio of the fluorescence signals at 460 nm and 530 nm reflects the level of TrkA activation.

[0276] Example 2. Development of TrkA antagonistic monoclonal antibodies After screening, the desired clones were subcloned and identified. These clones bound to human TrkA with high specificity and strength, and also showed strong inhibition in the TrkA-dependent NFAT antagonist reporter assay. As shown in the ELISA assay results (Figure 1) and FACS analysis results (Figure 2), the selected clones muPHD50, muPHD48, and muPHD49 showed desirable binding to human TrkA. Furthermore, as shown in the antagonist NFAT assay (Figure 3A and 3B), the selected antibodies muPHD31, muPHD50, muPHD48, and muPHD49 can inhibit the activation of TrkA signaling induced by human NGF.

[0277] Example 3. Cloning of antibody variable region genes Hybridoma cells were cultured in 10 cm dishes and harvested during logarithmic growth phase. Total cellular RNA was extracted using Trizol (Invitrogen, 15596-018) according to the manufacturer's instructions. RNA was resuspended in nuclease-free water. RNA concentration was measured using absorbance at 260 nm in a bioTEK instrument. To obtain cDNA templates, 4 μg of each RNA was reverse transcribed using a HiFiScript cDNA synthesis kit (CWBIO, CW2569). Then, PCR reactions were performed twice to clone the variable genes of the antibodies. The PCR products were directly sequenced. To construct expression plasmids for the chimeric antibodies, the VH genes were subcloned into the pCNDA3.4-hIgG4 vector and the VL genes were subcloned into the pCDNA3.4-hKappa vector, and the sequences were further confirmed by DNA sequencing. The complementarity determining regions (CDRs) of the antibodies were identified according to the Kabat, Enhanced Chothia, and IMGT systems (Dunbar J et al., Bioinformatics. 2016, 32(2):298-300).

[0278] The heavy and light chain variable region sequences of muPHD48 are shown in SEQ ID NOs: 16 and 18, respectively.

[0279] The CDRs of muPHD48 are as follows:

[0280] [Table 4] The heavy and light chain variable region sequences of PHD49 are shown in SEQ ID NOs: 42 and 44, respectively.

[0281] The CDRs of muPHD49 are as follows:

[0282] [Table 5] The heavy and light chain variable region sequences of muPHD50 are shown in SEQ ID NOs:51 and 53, respectively.

[0283] The CDRs for muPHD50 are:

[0284] [Table 6] The heavy and light chain variable region sequences of muPHD31 are shown in SEQ ID NOs: 14 and 15, respectively.

[0285] The CDRs of muPHD31 are as follows:

[0286] [Table 7]

[0287] Example 4. Humanization Humanization of the murine antibody was performed using CDR grafting as previously reported. Briefly, the parental (murine antibody) variable region (VH and VL) frameworks were replaced with the VH and VL frameworks of selected human germline V and J genes. The germline genes were selected based on the homology between the parental antibody and the germline V and J genes. Human HC germline genes IGHV1-46*01 and IGHJ3*01 were selected as FR donors for humanization of muPHD48 VH, and human LC germline genes IGKV1-39*01 and IGKJ4*01 were selected as FR donors for humanization of muPHD48 VL. The human HC germline genes IGHV1-69*01 and IGHJ5*01 were selected as FR donors for humanization of muPHD49 VH, and the human LC germline genes IGKV1-39*01 and IGKJ2*01 were selected as FR donors for humanization of muPHD49 VL.

[0288] The VH and VL sequences of humanized muPHD48 (i.e., PHD48-01) are as follows: PHD48-01 VH QVQLVQSGAEVKKPGASVKVSCKASGYSFTTYWMHWVRQAPGQGLEWIGTIYPGNSDSSNNQKFKGRATLTADTSTSTAYMELSSLRSEDTAVYYCTRFYYEDWYFDVWGQGTMVTVSS (SEQ ID NO: 89) PHD48-01 VL DIQMTQSPSSLSASVGDRVTITCSASSSVSYMYWFQQKPGKAPKPWIYRTSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYHSYPPTFGGGTKVEIK (SEQ ID NO: 90)

[0289] The VH and VL sequences of humanized muPHD49 (i.e., PHD49-01) are as follows: PHD49-01 VH QVQLVQSGAEVKKPGSSVKVSCKASGYTFSSYWMQWVRQAPGQGLEWIGAIYPGDDDTIYTQKFKGRATLTADKSTSTAYMELSSLRSEDTAVYYCARNYDYQAWFAYWGQGTLVTVSS (SEQ ID NO: 99) PHD49-01 VL DIQMTQSPSSLSASVGDRVTITCSASSSVSYMYWYQQKPGKAPKPWIYLTSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPLTFGQGTKLEIK (SEQ ID NO: 100)

[0290] The corresponding encoding nucleic acid sequences were synthesized and subcloned into pCDNA3.4-hIgG4 or pCDNA3.4-hKappa vectors, respectively, and then sequenced (GeneWiz). The expression plasmids for PHD48-01 and PHD49-01 consist of a humanized VH fused to a human IgG4 constant region and a humanized VL fused to a human Ig kappa constant region. All recombinant antibodies were expressed and purified as described in Example 1.

[0291] As shown in Figures 4A and 4B, the EC50 value of PHD48-01 in FACS analysis was approximately 142 ng / mL, comparable to 112 ng / mL for chi-PHD48 (chimeric antibody derived from muPHD48), and the IC50 value of PHD48-01 in NFAT antagonism assay was approximately 345 pM, comparable to muPHD48 (411 pM). These results suggest that PHD48-01 fully retains the binding and biological activity of muPHD48 and chi-PHD48.

[0292] As shown in Figures 5A and 5B, PHD49-01 retained similar functions to chi-PHD49. The EC50 value of PHD49-01 in FACS analysis was approximately 198 ng / mL, comparable to that of chi-PHD49 (157 ng / mL). The IC50 value of PHD49-01 in NFAT assay was approximately 604 pM, comparable to that of muPHD49 (404 pM).

[0293] Biolayer interferometry (BLI) with Octet Red384 was used to determine antibody affinity. Briefly, anti-human Fc coated biosensor AHQ chips (ForteBio) were soaked in PBS containing 0.1% w / v bovine serum albumin and 0.05% Tween-20 for a minimum of 10 minutes in a pre-wetted plate. Purified antibodies (100 nM in PBS containing 0.1% bovine serum albumin and 0.05% Tween-20 (assay buffer)) were captured on the AHQ biosensor (ForteBio) at a level of approximately 1 nm. The loaded biosensor was washed with assay buffer to remove unbound proteins. Binding rates, dissociation rates, and responses were then measured using 100 nM antigen (in PBS containing 0.1% bovine serum albumin and 0.05% Tween-20). The results are shown in the table below.

[0294] [Table 8] The PHD48-01 antibody showed affinity similar to that of the chimeric antibody. On the other hand, the PHD49-01 antibody showed lower affinity than the chimeric antibody, but still showed K D As shown by the values, the affinity was comparable to that of the chimeric antibody.

[0295] Two further rounds of humanization were performed. In the first round, fewer mouse amino acid residues were mutated while maintaining a high affinity comparable to the chimeric antibody. In the second round, many other variants (including some mutations in the CDR sequences) were further constructed. Quick-change-based site-directed mutagenesis or overlap PCR were performed to create expression plasmids for new versions of the humanized antibodies in the second round. All recombinant antibodies were expressed and purified as described in Example 1, and then their antigen-binding affinity was measured using BLI technology using the Octet Red384 instrument described above. NFAT assays were also performed to compare the functional activity of the humanized and chimeric antibodies. The results are summarized in the table below.

[0296] [Table 9]

[0297] Example 5. Affinity Maturation Antibody affinity maturation was performed by phage display as previously reported (Thie H et.al, Methods Mol Biol. 2009, 525:309-xv; Bostrom J et.al., Methods Mol Biol. 2009, 525:353-xiii). Random mutations were inserted by error-prone PCR using the GeneMorph II Random Mutagenesis Kit (Stratagene) according to the manufacturer's instructions. The mutated PCR products and the phage display vector were digested with NcoI-HF (NEB) and EcoRI (NEB) at 37°C overnight. Completely digested DNA was purified using the Axygen® AxyPrep PCR Cleanup Kit, and the respective DNA concentrations of the vector and PCR products were determined by measuring the absorbance at 260 nm. Ligations were then performed in a volume of 500 μL to achieve a vector:insert molar ratio of 1:3 and incubated at 16°C overnight. The ligation products were then desalted using AxYGen® AxYPrep PCR Cleanup Kit and introduced into XL1-blue electrotransformation high efficiency cells. The transformed cells were cultured in 15 mL of pre-warmed 2X YT medium at 37°C and 250 rpm for 2 hours. The transformation efficiency was calculated by plating an aliquot of each dilution after overnight incubation at 30°C. The remaining cell suspension was plated on 15 x 15 cm 2xYT agar plates. The next day, the cells were carefully scraped off from the plates with a spatula. High affinity clones were selected and sequenced.

[0298] The affinities of the antibodies are summarized in the table below.

[0299] [Table 10]

[0300] Example 6. Measurement of affinity by surface plasmon resonance (SPR) The binding kinetics and affinity of the humanized antibodies were determined using BIAcore T200 TM The results were determined using an SPR system. Approximately 30 RU of humanized antibody was immobilized on a protein G sensor chip, and serial 2-fold dilutions of PHA (0.625-20 nM) in 1xHBS-EP+ buffer were injected over the antibody-bound surface at a flow rate of 30 μL / min. The results are summarized as follows:

[0301] [Table 11]

[0302] Example 7. In vitro functional analysis of TrkA antibodies To assess the activity of the humanized TrkA antibodies of the invention, inhibition of NGF-induced TrkA activation was assessed using CellSensor® TrkA-NFAT-bla CHO-K1 cells. Cellsensor TrkA-NFAT-bla CHO-K1 cells (K1516, Life Technologies) were seeded in 96-well plates with 100 μL of medium the day before the experiment. Cells were incubated at 37° C. / 5% CO 2 The plates were pre-incubated with 3-fold serial dilutions of antibodies for 30 min in a humidified incubator at 37°C. Cell supernatants were then removed and 50 μL / well of 2×NGF was added. The plates were then placed in a humidified incubator at 37°C / 5% CO 2Incubated for 30 min in an incubator. Cell supernatant was carefully removed by aspirating the supernatant or by tapping the plate. 50 μL of supplemented lysis buffer (1X) was immediately added and incubated for 30 min at room temperature with shaking. After homogenization by pipetting, 16 μL of cell lysate was transferred from the 96-well plate to the 384-well plate. 16 μL of supplemented lysis buffer (1X) was dispensed into cell-free control wells. 16 μL of homogenized cell lysate was dispensed into test sample wells, unstimulated control wells, and stimulated control wells and incubated at room temperature for at least 4 h. Eu 3+ A CrYptate reader was set up and the fluorescence emission at two different wavelengths (665 nm and 620 nm) was read on a compatible HTRF® reader. NGF dose-response curves were analyzed with a four-parameter fit using GraphPad Prism software. The results are summarized in the table below.

[0303] [Table 12]

[0304] Example 8. Determination of the binding epitope of anti-TrkA antibody Full-length PHD48 antibody was digested with pepsin at a molar ratio of 1:200 (pepsin:antibody protein) for 240 min at 37 °C. 2-mercaptoethylamine HCl (2-MEA) at a final concentration of 50 mM was added to the sample and the sample was incubated at 37 °C for 30 min. PHD48 Fab was then purified by size-exclusion column. TrkA(ECD)-PHD48 Fab-anti-human Fab VHH (molar ratio 1:2:6) was prepared by incubating the three components on ice for 2 h.

[0305] Cryo-EM data were collected using a 300 kV Titan Krios electron microscope (Thermo Fisher Scientific, USA) equipped with a K3 direct electron detector (Gatan, USA) operated in counting mode. All movies were automatically recorded using EPU at a magnification of 105K and a physical pixel size of 0.819 Å. A total dose of 55.8 e- / Å2 was split into 40 frames. All image processing was performed using cryoSPARC v3.3.1, and patch CTF estimation, 2D classification, heterogeneous refinement, and homogeneous refinement were all performed in cryoSPARC. A total of 61,777,707 particles were automatically selected using a template picker and extracted with 2 × 2 binning (box size of 180 pixels, 1.638 Å per pixel). To select a dataset of good particles, 2D classification was performed twice. The selected particles were then subjected to heterogeneous refinement. The refined coordinates were used to re-center and re-extract the unbinned particles (0.819 Å per pixel, box size of 360 pixels), which were further refined to uniformity, resulting in a final density map of 3.11 Å.

[0306] To build a model of the TrkA(ECD)-PHD48 Fab-anti-human Fab VHH complex, the structures of TrkA(ECD) (from AlphaFold PDB) and PHD48 Fab (with Frizzled-5 protein removed from PDB 6WW2) were fitted to the cryo-EM map using UCSF Chimera (UCSF Chimera Home Page). The model was built manually in Coot (Emsley et al., 2010) with guidance from the cryo-EM map combined with real-space refinement using CCPEM.

[0307] The interaction of TrkA(ECD) with PHD48 was analyzed by UCSF Chimera. As shown in FIG. 6. The antigen-antibody interaction is mainly mediated by hydrogen bonds, and the interacting regions are residues 176 (Q), 178-180 (HGQ), and 187 (P) of TrkA(ECD), residues 33 (W), 35 (H), and 104 (W) of the PHD48 heavy chain (whose VH has the amino acid sequence set forth in SEQ ID NO: 86), and residues 31 (Y) and 90 (Y) of the PHD48 light chain (whose VL has the amino acid sequence set forth in SEQ ID NO: 91, Y31 corresponds to Y32 according to the Kabat numbering system in FIG. 6, and Y90 corresponds to Y91 according to the Kabat numbering system in FIG. 6). The interacting residue sites are shown in the following table.

[0308] [Table 13]

[0309] Example 9. Determination of affinity and function of TrkA antibody mutants The affinities of additional TrkA antibody variants were determined by a biolayer interferometry (BLI)-based method (Fortebio). All antibody variants were prepared as described in Examples 1 and 4 above.

[0310] The amino acid sequences of the TrkA antibody mutants are shown below.

[0311] [Table 14] The CDRs within the VH of PHD22 are as follows:

[0312] [Table 15] The CDRs within the VH of PHD24 are as follows:

[0313] [Table 16] The CDRs within the VH of PHD25 are as follows:

[0314] [Table 17] The CDRs within the VH of PHD30 are as follows:

[0315] [Table 18] The CDRs in the VL of PHD25 are as follows:

[0316] [Table 19] The CDRs within the VH of PHD26 are as follows:

[0317] [Table 20] The CDRs in the VL of PHD30 are as follows:

[0318] [Table 21] The affinity data is shown in the following table.

[0319] [Table 22] Furthermore, as shown in the antagonist NFAT assay (Figure 9), the TrkA antibodies PHD22, PHD24, PHD25, PHD26, PHD28, PHD29, PHD30, and PHD48 can inhibit the activation of TrkA signaling induced by human NGF, and their inhibitory activity is significantly superior to that of the control antibody pAb01.

[0320] Example 10. TrkA antibody competition assay To determine whether TrkA antibodies compete with each other for binding to the target. A biolayer interferometry (BLI)-based competition assay was performed for Octet Red384. Briefly, anti-pentaHIS (HIS1K) biosensors (Fortebio) were soaked in PBS containing 0.1% w / v bovine serum albumin and 0.05% Tween-20 (assay buffer) for a minimum of 10 min in a pre-wetted plate. 100 nM of TrkA ECD protein in assay buffer was captured onto the sensor, yielding a capture level of 0.3–1 nm. The loaded biosensor was then equilibrated with assay buffer for a minimum of 30 s, followed by binding of the first antibody for 90 s. After a second equilibration, the second antibody was loaded for 90 s. Antibodies PHD22, PHD24, PHD25, PHD26, PHD28, PHD29, PHD30, and PHD48 compete with each other for binding to TrkA. The control antibody pAb01 (having a VH set forth in SEQ ID NO:123 and a VL set forth in SEQ ID NO:124) does not compete with PHD22, PHD24, PHD25, PHD26, PHD28, PHD29, PHD30, or PHD48 (Figures 7A-7D).

[0321] Example 11. NGF Inhibition Assay To determine whether TrkA antibodies can inhibit the binding between TrkA and NGF. A biolayer interferometry (BLI)-based competition assay was performed against Octet Red384. Briefly, anti-human Fc (AHC) biosensors (Fortebio) were soaked in PBS containing 0.1% w / v bovine serum albumin and 0.05% Tween-20 (assay buffer) for a minimum of 10 min in a pre-wetted plate. 100 nM of antibody in assay buffer was captured onto the sensor, yielding a capture level of 0.5–1.5 nm. The loaded biosensor was then equilibrated in assay buffer for a minimum of 15 s, followed by binding of TrkA ECD for a minimum of 60 s. After a second equilibration, NGF was loaded for a minimum of 60 s. Antibodies PHD22, PHD24, PHD25, PHD26, PHD28, PHD29, PHD30, PHD48, and PHD49 do not block the binding between TrkA and NGF. In contrast, the control antibody pAb01 inhibits the binding between TrkA and NGF (Figures 8A-8C).

[0322] Example 12. Effect of TrkA antibodies on survival and axonal outgrowth of DRG neurons DRG isolated primary neurons were prepared from humanized fetal mice. DRGs were harvested, incubated with 0.05% trypsin-EDTA for 5–8 min at 37 °C, pipetted into single cells, and plated at a density of 5000 cells / well on 24-well plates containing different DRG-conditioned media (NGF 0 nM, NGF 0.3 nM, NGF 0.3 nM / PHD48 3 nM, NGF 0.3 nM / tanezumab 3 nM, NGF 0.3 nM / fasinumab 3 nM) in poly-D-lysine and laminin-pretreated Petri dishes. Half of the DRG-conditioned media was replaced after 48 h, and 5-fluoro-2'-deoxyuridine was added at a final concentration of 5 μg / mL to suppress excessive proliferation of glial cells. 72 h after DRG seeding, 10x and 20x bright-field imaging was performed. Bright-field photographs showed normal survival and axonal extension of DRG neurons in the NGF 0.3 nM group and the NGF 0.3 nM / PHD48 3 nM group, as shown in Figure 10. In the NGF 0 nM group, the tanezumab 3 nM group, and the NGF 0.3 nM / fasinumab group, only a few DRG neurons survived, and the axons of the surviving neurons were extremely short.

[0323] 72 hours after DRG seeding, the mid-field of each well in the experimental group was photographed (20x), the axonal pathways of DRG neurons were traced and their length was calculated with ImageJ, and the average length of each field was used for the above statistics. Graphs were generated using GraphPad software, and data were reported as mean ± standard error of the mean (SEM). One-way ANOVA was used to analyze the data. ****P<0.0001. As shown in Figure 11, the axonal length of the NGF 0.3nM group and the NGF 0.3nM / PHD48 3nM group was over 400μm, and there was no significant difference in the axonal length between the two groups. The mean axon lengths of the 0 nM NGF group, the 0.3 nM / 3 nM NGF tanezumab group, and the 0.3 nM / 3 nM NGF fasinumab group were all less than 100 μm, and the axon lengths of DRG neurons in the 0.3 nM / 3 nM NGF fasinumab group were significantly shorter. These results indicate that PHD48, unlike the NGF neutralizing antibodies (tanezumab and fasinumab), did not exert a significant inhibitory effect on NGF-mediated survival and axon extension of DRG neurons.

[0324] Example 13. TrkA antibodies reduce NGF-induced pain in mice Inflammation and neuronal damage result in an early and sustained increase in NGF levels in vivo. In adults, NGF can increase sensitivity to noxious stimuli and cause hyperalgesia. Injection of exogenous NGF produces changes in mechanical and thermal hypersensitivity in rodents and humans. Intraplantar injection of NGF produces a relatively short-lived increase in mechanical hypersensitivity.

[0325] To understand the antinociceptive effects of the TrkA antibodies of the present invention (e.g., PHD48), the NGF-induced hypersensitivity model (by intraplantar injection of NGF) was used as a model (Figure 12). The results demonstrated that subcutaneous injection of fasinumab significantly attenuated heat hypersensitivity (24 hours after NGF injection, Figure 13A) and mechanical hypersensitivity (72 hours to 120 hours after NGF injection, Figure 13B), indicating that the hypersensitivity is mediated by the NGF signaling pathway. In this model, PHD48 also significantly attenuated heat hypersensitivity (24 hours after NGF induction, Figure 13A) and mechanical hypersensitivity (48 hours to 120 hours after NGF injection, Figure 13B) by inhibiting NGF / TrkA signaling.

[0326] Example 14. TrkA antibodies reduce formalin-induced pain in mice Nerve growth factor (NGF) exerts potent analgesic effects on the peripheral nervous system primarily via its receptor TrkA.

[0327] The formalin test in mice is a reliable model of pain sensation and is sensitive to various types of analgesics. The noxious stimulus was the injection of dilute formalin under the dorsal skin of the right hind paw. Pain behavior was recorded as the number of movements (paw licking) from 0 to 45 min after formalin injection. Then, the total number of movements during the late phase (20 to 30 min after formalin injection), which represents inflammatory pain, was calculated and analyzed among different groups.

[0328] To understand the antinociceptive effect of the TrkA antibody (e.g., PHD48) of the present invention, a formalin-induced inflammatory pain mouse model was established (Figure 14), and it was found that subcutaneous injection of fasinumab significantly alleviated the formalin-mediated increase in motor counts (Figures 15A and 15B). This indicates that formalin-induced pain is mediated by the NGF signal pathway. Meanwhile, PHD48 also significantly alleviated the formalin-mediated increase in motor counts (Figures 15A and 15B). This indicates that the TrkA antibody of the present disclosure is effective in this pain model.

[0329] Example 15. TrkA antibody improved MIA-induced OA pain in mice Monoiodoacetic acid (MIA) injection into the knee joint causes progressive destruction of cartilage, which leads to the development of pain-like behaviors. The pathological changes induced by MIA have many similarities with those observed in human OA, such as cartilage loss and subchondral bone changes.

[0330] To study the effect of the TrkA antibody of the present invention (e.g., PHD48) on OA pain, MIA was injected into the knee joint of mice to induce the OA pain model (Figure 16). Except for the control group, animals in other groups were injected with MIA into the knee joint, and baseline screening was performed 9 days after MIA injection. Mice that met the model criteria (paw withdrawal threshold < 0.6g) in each group were selected and administered the test drug. Celecoxib (TCI, Lot No.: CJDBF-RG) was orally administered at 100 mg / kg as a positive control, and significantly reduced the mechanical hypersensitivity of MIA-treated mice (Figures 17A-17G). Subcutaneous injection of fasinumab also significantly suppressed the mechanical hypersensitivity of MIA-treated mice (Figures 17A-17G). This indicates that MIA-induced pain is mediated by the NGF signaling pathway. On the other hand, treatment with the TrkA antibody of the present disclosure (e.g., PHD48) significantly alleviated mechanical hypersensitivity in MIA-treated mice (Figures 17A-17G), indicating that the TrkA antibody of the present disclosure exhibited promising antinociceptive effects for the treatment of OA pain.

[0331] Although preferred embodiments of the present application have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention be limited by the specific examples provided herein. Although the present invention has been described with reference to the above description, the description and drawings of the embodiments herein are not meant to be interpreted in a limiting sense. Various modifications, changes, and substitutions will occur to those skilled in the art without departing from the present invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions described herein, which depend on a variety of conditions and variables. It should be understood that various alternatives can be used in the embodiments of the present invention described herein in the practice of the present invention. It is therefore contemplated that the present invention must also include any such alternatives, modifications, variations, and equivalents. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of these claims, and equivalents thereof, are covered by the claims.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to TrkA and has one or more properties selected from the group consisting of: 1) When measured by Octet or SPR, it is approximately 5*10 －８ K less than M Ｄ capable of binding to TrkA at 2) the ability to inhibit NGF-induced TrkA activation; 3) does not substantially inhibit the binding between TrkA and NGF; 4) does not substantially compete with NGF for binding to TrkA; and 5) It is possible to selectively alleviate NGF-mediated pain sensitization without substantially impairing the effects of NGF on neuronal growth and survival. Showing, An antibody or antigen-binding fragment thereof.

2. It is capable of recognizing an epitope in the TrkA extracellular domain (ECD), wherein the epitope comprises amino acid residues Q176, H178, G179, Q180, and P187 of SEQ ID NO: 119; The antibody or antigen-binding fragment thereof according to claim 1.

3. capable of specifically binding to Q176, H178, G179, Q180 and / or P187 of SEQ ID NO: 119; The antibody or antigen-binding fragment thereof according to claim 2.

4. the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, a fully human antibody, and a multispecific antibody; the antigen-binding fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab)2 fragment, an Fv fragment, a VHH, and an ScFv; The antibody or antigen-binding fragment thereof according to claim 1.

5. the TrkA is human TrkA; The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

6. can compete with a reference antibody for binding to TrkA, wherein the reference antibody comprises light chain CDR1-3 and heavy chain CDR1-3, wherein the light chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 96-98, respectively, and the heavy chain CDR1-3 comprise the amino acid sequences set forth in SEQ ID NOs: 93-95, respectively; The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

7. the light chain CDR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 96, 117, 9, and 50; the light chain CDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 97, 118, and 11; and the light chain CDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 98, 13, and 41; The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

8. the light chain CDR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 9, 28, 50, 56, 80, and 88; the light chain CDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 11, 30, 39, 58, 77, 82, 103, and 108; and the light chain CDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 13, 32, 41, and 60; The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

9. a light chain variable region, wherein the light chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 132, 134, 15, and 53; The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

10. a light chain variable region, wherein the light chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 15, 18, 44, 53, 61, 78, 85, 90, 91, 100, 104, 107, and 113; The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

11. a light chain constant region, wherein the light chain constant region comprises a human Igκ constant region or a human Igλ constant region; The antibody or antigen-binding fragment thereof described in claim 7.

12. heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, wherein the heavy chain CDR1 is selected from the group consisting of SEQ ID NOs: 93, 114 (SX １ WX ２ Q, here, X １ is H or Y, and X ２ wherein SEQ ID NO: 1 is I or M), 1, and 47, wherein the heavy chain CDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 94, 115, 4, and 48, and the heavy chain CDR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 95, 116, 7, and 49; The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

13. the heavy chain CDR1 comprises a heavy chain CDR2 and a heavy chain CDR3, wherein the heavy chain CDR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1, 20, 45, 47, 62, 101, and 109, the heavy chain CDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 4, 23, 46, 48, 65, 73, 75, 87, 102, and 110, and the heavy chain CDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 7, 26, 37, 49, 68, and 111; The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

14. a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 131, 133, 14, and 51; The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

15. a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 14, 16, 42, 51, 70, 74, 76, 84, 86, 89, 92, 99, 105, 106, and 112; The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

16. a heavy chain constant region, wherein the heavy chain constant region comprises a human IgG constant region; The antibody or antigen-binding fragment thereof described in claim 12.

17. 1) light chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 96, 97, and 98, respectively, and heavy chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 93, 94, and 95, respectively; 2) light chain CDRs 1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 117, 118, and 41, respectively, and SEQ ID NO: 114 (SX １ WX ２ Q, here X １ is H or Y, and X ２ 115, and 116, respectively; 3) light chain CDRs 1-3 comprising the amino acid sequences set forth in SEQ ID NOS: 9, 11, and 13, respectively, and heavy chain CDRs 1-3 comprising the amino acid sequences set forth in SEQ ID NOS: 1, 4, and 7, respectively; 4) light chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 28, 30, and 32, respectively, and heavy chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 20, 23, and 26, respectively; 5) light chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 56, 58, and 60, respectively, and heavy chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 62, 65, and 68, respectively; 6) light chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 56, 58, and 60, respectively, and heavy chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 20, 73, and 68, respectively; 7) light chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 56, 58, and 60, respectively, and heavy chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 20, 75, and 68, respectively; 8) light chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 56, 77, and 60, respectively, and heavy chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 62, 65, and 68, respectively; 9) light chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 56, 77, and 60, respectively, and heavy chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 20, 73, and 68, respectively; 10) light chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 56, 77, and 60, respectively, and heavy chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 20, 75, and 68, respectively; 11) light chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 80, 82, and 32, respectively, and heavy chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 62, 79, and 26, respectively; 12) light chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 88, 30, and 32, respectively, and heavy chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 20, 87, and 26, respectively; 13) light chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 28, 39, and 41, respectively, and heavy chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 37, respectively; 14) light chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 88, 103, and 41, respectively, and heavy chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 101, 102, and 37, respectively; 15) light chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 88, 103, and 41, respectively, and heavy chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 109, 110, and 111, respectively; 16) light chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 88, 39, and 41, respectively, and heavy chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 37, respectively; 17) light chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 39, 108, and 41, respectively, and heavy chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 109, 110, and 111, respectively; or 18) light chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 50, 39, and 41, respectively, and heavy chain CDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 47, 48, and 49, respectively; Including, The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

18. 1) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 132, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 131; 2) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 134, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 133; 3) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 15, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 14; 4) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 18, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16; 5) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 90, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 89; 6) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 91, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 86; 7) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 91, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 92; 8) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 61, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70; 9) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 61, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 74; 10) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 61, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 76; 11) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 78, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70; 12) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 78, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 74; 13) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 78, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 76; 14) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 85, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 84; 15) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 44, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 42; 16) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 100, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 99; 17) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 104, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 105; 18) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 104, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 112; 19) A light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 107, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 106; 20) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 113, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 112; or 21) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 53, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 51; Including, The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

19. A fusion protein comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

20. A protein complex comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

21. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

22. A vector comprising the isolated nucleic acid molecule of claim 21.

23. 22. A cell comprising the isolated nucleic acid molecule of claim 21.

24. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and a pharmaceutically acceptable excipient.

25. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described in any one of claims 1 to 3 for use in the prevention and / or treatment of a disease or disorder associated with inappropriate expression or function of TrkA.

26. 26. The pharmaceutical composition of claim 25, wherein the disease or disorder comprises pain.

27. 27. The pharmaceutical composition of claim 26, wherein the pain comprises chronic pain.

28. the disease or disorder comprises chronic pain of nociceptive, inflammatory, neuropathic, proliferative or mixed etiology; 28. The pharmaceutical composition of claim 27.

29. The disease or disorder includes chronic pain caused by musculoskeletal or neurological disorders.

27. The pharmaceutical composition of claim 26.

30. The disease or disorder comprises post-operative pain, rheumatoid arthritis pain, neuropathic pain and / or osteoarthritis pain; 27. The pharmaceutical composition of claim 26.

31. Use of an epitope, comprising: Use of an epitope of the TrkA extracellular domain (ECD) in the manufacture of an agent for obtaining or screening a TrkA antibody that does not substantially inhibit the binding of TrkA to NGF, wherein the epitope comprises amino acid residues Q176, H178, G179, Q180, and P187 of SEQ ID NO:

119.

32. The TrkA antibody has one or more properties selected from the group consisting of: 1) When measured by Octet or SPR, it is approximately 5*10 －８ K less than M Ｄ capable of binding to TrkA at 2) the ability to inhibit NGF-induced TrkA activation; 3) does not substantially inhibit the binding between TrkA and NGF; 4) does not substantially compete with NGF for binding to TrkA; and 5) It is possible to selectively alleviate NGF-mediated pain sensitization without substantially impairing the effects of NGF on neuronal growth and survival. Showing, 32. The use according to claim 31.

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